ENZYMES AND THE EFFECT OF TEMPERATURE

ABSTRACT
In this experiment, factors that affect the invertase is also investigated like the effect of temperature .The results
obtained were represented by bell-shaped graphs that shows the optimum amount of temperature. Although, the
expected shape was not achieved because activation energy is spontaneous therefore the transition state was inferred to the highest
value of absorbance in the graph. The highest peak of the curve represents the highest concentration which was determined by the
Absorbance. Temperature above 60C shows that the invertase reached denaturation.

INTRODUCTION
Living organisms are composed of complex
systems of chemical reactions. In the absence of
catalysis, most reactions in biological systems
would take place far too slowly to provide
products at an adequate pace for a metabolizing
organism. The catalysts that serve this function
in organisms are called enzymes [1]. Enzymes
accelerate the reactions without being changed
themselves. They have three characteristics.
First, its basic function is to speed up the rate of
a reaction. Second, most enzymes act specifically
with one reactant called a substrate to produce
products. Lastly, enzymes are regulated from a
state of low activity to high activity and vice
versa [2].
Sucrose is common as table sugar is a
frequently occurring disaccharide composed of D-glucose and -D-fructose molecules linked by
an -1,4-glycosidic bond. It is hydrolyzed to an
equimolar mixture of glucose and fructose. Called
invert sugar, this mixture of monosaccharides is
derived from the fact that sucrose rotates that
plane
of
polarized
light
to
the
right
(dextrorotatory) however hydrolysis products
rotate the plane of polarized light to the left
(levorotatory) [1].
Sucrose can be hydrolyzed by an enzyme called
invertase (Equation 1).

Sucrose + H 2 O Invertase DGluc ose + DFrustose

Equation 1. Hydrolysis of Sucrose catalyzed by

enzyme Invertase
Invertase, usually found in plants, acts as a
catalyst for the hydrolysis of sucrose. Its
significance as an enzyme lies on the importance
of glucose as an important product of
photosynthesis. In the confectionery industry
where fructose, on the other hand is preferred
rather than sucrose because it is sweeter and not
crystallized easily [4].

Dinitrosalicylic acid (D.N.S.A. or 3,5dinitrosalicylic acid) is a reagent used in

determining sugar content. DNS technique is
utilized in order to estimate sugar present in the
blood, in the cerebrospinal fluid, and other
human body liquids. The amount of sugar in the
blood has metabolic implications used to
determine blood sugar related diseases which
make the use of DNS a good way to assess sugar
level in the blood [2].

Figure
1.
Chemical
structure
of
3,5dinitrosalicylic acid
Since enzymes are proteins, they are
denatured by elevated temperature. Denaturation
is the process of unfolding of a protein (i.e.,
disruption of the tertiary structure. On the other
hand reduction of disulfide bonds leads to even
more extensive unraveling of the tertiary
structure [1].All enzymes are functionally specific
to varying degrees. Enzyme activity is the rate of
the catalyzed reaction. When plotted against
temperature, the curve usually has a peak or
optimum temperature. The region for optimum
temperature does not inescapably mean it has to
be at the exact value for effective catalysis.

heated in their respective temperatures for 5

minutes. All of the test tubes were added 1.5 ml
of sucrose solution and heated again for 5
minutes. Finally adding 3 ml of Dinitrosalicyclic
reagent and heating all of the reagents at 95C
for 5 minutes. The solutions were subjected to
spectrophotometer at 560nm molar absorbance.

Figure 2. Effect of temperature on enzyme rate

of reaction
Figure 2 shows the effect of temperature on
the activity of an enzyme catalyzed reaction [4].
In explaining the bell-curve trend seen above,
in general, enzymes are preferably used at high
temperatures in order to make use of its
increased rate of reaction plus the protection it
provides from microbial infection. Nevertheless,
enzymes are proteins and they undergo
essentially
irreversible
denaturation
at
temperatures above those to which they are
ordinarily exposed in their natural environment.
This implies that at a critical temperature, there
is a rapid rate of loss of activity [3].
Experimental
A. Compounds Tested
Bakers Yeast,
Buffer solution (pH 5)
B. Procedure
1. Extraction of Invertase from yeast
Bakers yeast weighing 0.25 grams was
dissolved in 150 ml distilled water. The solution
was allowed to stand for 20 minutes at 37C.
Supernatant was collected when sedimentation
occurred. The supernatant serves as the enzyme
stock solution.
2. Preparation of denatured invertase
stock solution
50 ml of the enzyme stock solution was placed
in a boiling water bath for 5 minutes. The
solution was allowed to cool. Supernatant was
collected when frothing occurred, the supernatant
served as the denatured enzyme stock solution.
3. Effect of temperature on invertase
activity.
Different temperatures of water bath set-ups
were prepared. See Table 1. All of the test tube
contains 2.9 ml buffer solution pH 5.00. Then
added 0.1 ml enzyme solution except for the
blank, 0.1 ml of denatured enzyme solution was
added to blank. After that the test tubes were

Test Tube No.

1
2
3
4
5
6
7 (blank)

Temperature
37C
40C
50C
60C
70C
90C
37C

Table 1: Test tube numbers

corresponding temperature.

Figure 3. Set-ups
temperatures

of

different

with

their

water

bath

RESULTS AND DISCUSSIONS

The temperature affects the invertase activity.
The optimum temperature of the invertase should
be at 60C (140F). A temperature too low will
have a slow reaction same as with high
temperatures
because
of
irreversible
denaturation.
Temperature

Absorbance

Blank

0.000

RT

0.032

40

0.372

50

0.778

60

0.784

70

0.814

90

0.899

Table 2. Effect of Temperature on Invertase

activity

Absorbance vs. Temperature

It points out that an enzymes activity is regulated and

only occurs to a limited extent in biological reactions.
REFERENCES

[1]Campbell M. et. Al. (2012).Biochemistry,

7th edition, International Edition. China: Mary
Finch

0.8
0.6

[2]http://www.elmhurst.edu/~chm/vchembo
ok/570enzymes.html Date retrieved: March
16, 2015

Absorbance 0.4
0.2
0
37 (Blank)

40

60

90

Temperature

Figure 4. Absorbance vs Temperature Graph

As observed Figure 4 above, the experiment did not
meet the expected bell-shaped curve. It infers that the
activation energy is spontaneous that the transition state
was inferred to the highest value of absorbance in the
graph. The highest peak of the curve represents the
highest concentration which was determined by the
Absorbance. Temperature above 60C shows that the
invertase undertook denaturation which should have been
illustrated by sloping down of the curve.

[3]http://www1.lsbu.ac.uk/water/enztech/te
mperature.html Date retrieved: March 16,
2015
[4] William Ward (2012). The Isolation of
Invertase
from Bakers Yeast An
Introduction
to
Protein
Purification
Strategies, Protein Purification, Dr. Rizwan
Ahmad (Ed.), ISBN: 978-953-307-831-1,
InTech,
Available
from:
http://www.intechopen.com/books/proteinpurification/the-isolation-of-invertase-frombaker-s-yeast-anintroduction-to-proteinpurification-strategies