ENZYMES AND THE EFFECT OF PH

ABSTRACT
In this experiment, factors that affect the invertase is also investigated like the effect of pH .The results obtained were
represented by bell-shaped graphs that shows the optimum amount of pH. The Invertase can be affected by the

changes in pH. An Extremely high or low pH values generally result in complete loss of activity for most
enzymes.
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.

Sucrose + H 2 O Invertase DGlucose + DFrustose

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 2. Chemical structure of 3,5-dinitrosalicylic

acid
Since enzymes are proteins, they are sensitive
to changes in pH. Each enzyme has a specific and
optimum pH wherein it is most active. The result
of the effect of pH on a combination of factors
includes: (a) enzyme-substrate binding, (b)
catalytic activity of the enzyme, (c) ionization of
the substrate, and (4) variation in protein
structure [1]. The rates for many enzymatic
reactions would exhibit a bell-shaped curve as a
function of pH as seen below.

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 [3].

Figure 1. Effect of pH on enzymatic activity

The best explanation for this type of curve is

the stability of the enzyme during pH alteration.
This would mean too low and too high pH would
give the same slow reaction rate. The peak on
the bell as shown above demonstrates the best
working pH suitable for the enzyme because it
reached the maximum reaction rate for the
enzyme activity.

Experimental
A. Compounds Tested
Bakers Yeast,
Buffer solutions (pH 2, 3, 4, 5, 7, 9, 11)
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 PH on invertase activity
Seven test tubes and a blank test
tube were prepared and labeled with the
pH of the appropriate buffer solution.
2.90 mL of the certain pH was pipetted
into the test tubes. For the blank test
tube it contains 2.90 ml of distilled
water. Table 1 shows the number of test
tubes with their appropriate pH. Then
add 0.1 ml of the enzyme solution to the
seven test tubes that contains buffer
solutions while add the denatured
enzyme to the blank test tube. After that
heat in water bath for 5 minutes at
60C. Next is the addition of 1.5 ml
sucrose solution to all of the test tubes
without removing it in 60C water bath
for 5 minutes. Then 3 mL of
Dinitrosalicyclic reagent was added. The
test tubes were immersed at 95C water
bath for 5 minutes. Then the absorbance
at 540 nm was measured. Finally, the
amount of sucrose hydrolyzed was
determined using hydrolyzed-sucrose

standard curve that was constructed in

the Dinitrosalicyclic colorimetric method.
Test tube
Number
1
2
3
4
5
6
7
8

pH
2.00
3.00
4.00
5.00
7.00
9.00
11.00
BLANK

Table 1: Number of test tubes with their

appropriate pH

RESULTS AND DISCUSSIONS

The Invertase can be affected by the changes
in pH. An Extremely high or low pH values
generally result in complete loss of activity for
most enzymes.
pH

Absorbance

blank

0.000 A

0.774 A

0.653 A

0.275 A

0.059 A

0.375 A

0.106 A

11

0.120 A

Table 3. Effect of pH on Invertase Activity

Figure 3: Graph of the effect of pH on invertase

activity

The most favorable pH value, which is the point

where the enzyme is most active, is known as
the optimum pH is at pH 2. It shows that at pH 3

and above it the concentration of invertase will slope

down indicating denaturation of enzyme. It indicates that
invertase reactivity is controlled and occurs to a limited
extent.
REFERENCES
[1]Campbell M. et. Al.(2012).Biochemistry, 7th
edition, International Edition. China: Mary Finch
[2]http://www.elmhurst.edu/~chm/vchembook/5
70enzymes.html
[3] William Ward (2012). The Isolation of
Invertase from Bakers Yeast An Introduction to

Protein
Purification
Strategies,
Protein
Purification, Dr. Rizwan Ahmad (Ed.), ISBN: 978953-307-831-1,
InTech,
Available
from:
http://www.intechopen.com/books/proteinpurification/the-isolation-of-invertase-frombaker-s-yeast-anintroduction-to-proteinpurification-strategies Date Retrieved: March 16,
2015