ENZYMES AND EFFECTS OF pH

Thiara Terri V. Bella, Terrence Louis P. Carlos, Joseph Bernard E. Cordova, Jose Alfonso P. Cuisia and
Crystel Mhariel V. Daroy
Group 2 2F Medical Technology General Biohemistry Laboratory

ABSTRACT
Enzymes are biological molecules(proteins) that act as a catalyst and help complex reactions occur everywhere in life.
Invertase is an enzyme that catalyzes the breakdown of sucrose to form glucose and fructose. Invertase was
extracted from Bakers yeast and the substrate was collected to determine the effects of changes in pH on the reaction
rates of an enzyme-catalyzed reaction. 2.90 ml of buffer was placed in 7 test tubes having a pH of 2,3,4,5,7,9, and
11. The enzyme stock solution was poured and incubated. At the end of the experiment, spectrophotometry was used
at 540nm to be able to measure its absorbance.

INTRODUCTION
Enzymes are complex proteins that cause a
specific chemical change in all parts of the body.
For example, they can help break down the foods
we eat so the body can use them. Blood clotting
is another example of enzymes at work.

sugars and other reducing molecules to form 3amino-5-nitrosalicylic acid, which absorbs light
strongly at 540 nm[3] thus the need to use
spectrophotometry to measure the solutions
absorbance.

Enzymes are needed for all body functions. They

are found in every organ and cell in the body,
including in the blood, intestinal fluids,
mouth(saliva), stomach (gastric juice). [1] Other
experimental factors that affect the enzyme
activity includes: enzyme concentration, pH of
the reaction solution, temperature, substrate
concentration, enzyme inhibitors/activators and
cofactors/coenzymes.
The experiment focused on the effect of pH on
the enzyme(invertase) activity. Invertase is an
enzyme
that
catalyzes
the
hydrolysis
(breakdown) of sucrose (table sugar).Alternate
names for invertase include EC 3.2.1.26,
saccharase, glucosucrase, beta-h-fructosidase,
beta-fructosidase, invertin, sucrase, maxinvert L
1000, fructosylinvertase, alkaline invertase, acid
invertase and the systematic name: betafructofuranosidase. The resulting mixture of
fructose and glucose is called inverted sugar
syrup.Invertase is a yeast derived enzyme. It is
also synthesized by bees, who use it to make
honey from nectar.[2]
Denaturation is the complete loss of organized
structure in a protein, it is irreversible and it also
involves disruption of the secondary and tertiary
structure
without breaking the primary
structure. In the preparation of the solution,
invertase was denatured at 95 degrees Celsius to
stop activation energy which enabled the group
to determine the distinct period.
3,5-Dinitrosalicylic acid (DNS or DNSA, IUPAC
name 2-hydroxy-3,5-dinitrobenzoic acid) is an
aromatic compound that reacts with reducing

Figure 1. Bell shape curve of effects of pH

on invertase activity
Enzymes are affected by changes in pH. The
most favorable pH value - the point where the
enzyme is most active - is known as the optimum
pH.
.

EXPERIMENTAL
A. Compounds tested
For extraction of invertase from yeast:
0.25 g of Bakers yeast and 150mL
distilled water.
For preparation of denatured invertase
stock solution: 100mL enzyme stock
solution, and boiling water bath.

For effect of pH on invertase activity: 2.90 mL

0.1 M appropriate buffer solution, enzyme stock
solution, 60 and 95 degrees Celsius water bath,
3.00mL DNS reagent, 1.50mL sucrose solution
and UV-Vis Spectrophotometer.
B. Procedure
1. Extraction of Invertase from Yeast
0.25 g of Bakers yeast was dissolved in 150mL
distilled water. It was agitated for 5 minutes was
incubated in a water bath with a temperature of
37 degrees Celsius. The solution was decanted
and filtered. The supernatant was collected and
served as the enzyme stock solution that will be
used for the succeeding experiments.

Figure 2. Prepared solutions with different

pH

RESULTS AND DISCUSSIONS

2. Preparation
of
Denatured
Invertase Stock Solution
100mL enzyme stock solution was incubated in
a boiling water bath for 10 minutes. The solution
was allowed to cool and the supernatant can only
be collected if frothing or formation of
overflowing mass of bubbles occurs. This serves
as the denatured enzyme stock solution that will
be used for the succeeding experiments.
3. Effect of pH on Invertase Activity
7 numbered test tubes was prepared and 1 test
tube for the blank solution. 2.90mL of buffer was
poured on every test tube with the pH 2, 3, 4, 5,
7, 9, and 11. 0.10 mL enzyme stock solution was
added to each solution, mixed thoroughly,
incubated for 60 degrees Celsius water bath for 5
minutes. 1.50mL of sucrose solution was added
and incubated again for 5 minutes in a 60
degrees Celsius water bath. 3mL of DNS reagent
was added then immersed in a 95 degrees
Celsius water bath for 5 minutes to develop the
characteristic red-brown color. The solutions was
allowed to cool. Blank solution was prepared by
following the steps mentioned but the denatured
enzyme was used instead of the enzyme stock
solution. Absorbance was measured at 540nm.
The amount of sucrose hydrolyzed was
determined using hydrolyzed-sucrose standard
curve
constructed
in
the
dinitrosalicylic
colorimetric method.

Figure 3. Structures of sucrose and the

cyclic (anomeric) conformations of each
monosaccharide
The figure above shows the activity of invertase
as it catalyzes the breakdown of sucrose into
glucose and fructose. After the extraction of
invertase from the Bakers yeast, it was used to
determine the effects of pH on the enyme
activity. In this experiment, the rate of reaction is
based on the amount of sucrose hydrolyzed by
the invertase. Consequently, the reaction rate
usually increases as the amount of sucrose
hydrolyzed by the invertase . Thus, the reaction
rate has a direct relationship to the amount of
sucrose hydrolyzed.
Invertase splits the disaccharide sucrose into
the monosaccharides glucose and fructose.
Invertase is inhibited by high concentrations of its
substrate, sucrose. The invertase we supply has
optimum activity at 60 C. Its optimum pH is 4.5
(the pH is usually adjusted to this level by the
addition of citric acid to the reaction mix),
although it is active between pH 3.0 and 5.5.
Inactivation of the enzyme begins at 65 C and
the enzyme is totally inactivated after 5 minutes
at 90 C.

direct relationship with the amount of sucrose

hydrolyzed(mg/mL). Table 3 would yield a chart
as seen in Figure 5 below.
Figure 5. Graph of pH vs. Absorbancej

Figure 4. Standard curve plotted from the

Colorimetric method and the amount of
sucrose hydrolyzed per minute as a function
of pH
In the sucrose assay using Dinitrosalicylic
Colorimetric method, absorbance was compared
to the amount of acid-hydrolyzed sucrose which
produced a standard curve that as absorbance
increases, the amount of hydrolyzed sucrose also
increases. As seen also in figure 4, the amount of
hydrolyzed sucrose was highest at around pH 45. Extremely high or low pH values generally
result in complete loss of activity for most
enzymes. pH is also a factor in the stability of
enzymes. As with activity, for each enzyme there
is also a region of pH optimal stability.This is the
pH wherein the enzyme invertase is most active
or what we call as the optimum pH
Table 3. Effect of pH on Invertase Activity
pH
2
3
4

Absorbance at 540nm
-0.004A
-0.012A
-0.011A

5
7
9
11

0.011A
-0.008A
0.004A
-0.002A

Sugar Assay using dinitrosalicylic colorimetric

method was not done in the experiment and
absorbance was used as a reference to know the
optimum pH of the invertase. Absorbance at
540nm can be used as a reference since it has a

As seen in the graph, the highest peak is at

pH4-5 which means this the optimum pH wherein
the enzyme invertase is most active hydrolyzing
also most of the sucrose. We cant use equimolar
concentrations of glucose and sucrose
as a
standard solution for the construction of standard
curve for it would not yield an upward slope
graph but instead the opposite. Some errors were
made during the experiment so the bell shaped
curve which is the correct curve of the graph was
not achieved. The curve doesnt show a definite
relationship between pH and the rate of invertase
activity for the invertase activity is only at its
peak at optimum pH.
In addition to temperature and pH there are
other factors, such as ionic strength, which can
affect the enzymatic reaction. Each of these
physical and chemical parameters must be
considered and optimized in order for an
enzymatic
reaction
to
be
accurate
and
reproducible.

REFERENCE:
[1] Retrieved from http://www.worthingtonbiochem.com/introbiochem/effectsph.html
on Mar. 17, 2015
[2] Retrived from
http://cdn.intechopen.com/pdfswm/26595.pdf on Mar. 17, 2015

[3] Retrieved from

http://www.nlm.nih.gov/medlineplus/ency/ar
ticle/002353.htm on Mar. 17,2015
[4] Miller, G.L. (1959). Use of dinitrosalicylic
acid reagent for determination of reducing
sugar. Analytical Chemistry 31(3), 426-428
[5] Wang, N.S. Experiment 14: Enzyme
kinetics of invertase via initial rate
determination.Retrieved from
http://www.eng.umd.edu on Mar. 17,2015
[6] Wang, N.S. Experiment 9D: Sucrose
assay by dinitrosalicylic acid colorimetric
method.Retrieved from
http://www.eng.umd.edu on Mar. 17,2015