Effect of pH on Invertase Activity and Sucrose Assay using DNS Method

Jennelie B. Manaog, Charisse V. Misola, Chrisha R. Montemayor, Hajime Q. Nakaegawa, Maria Elijah A. Natanawan
Group 5, 2A Biochemistry, Faculty of Pharmacy, University of Santo Thomas

ABSTRACT
Enzymes are biomolecules that speed up or catalyze chemical reactions by lowering the energy of activation. This
experiment involves the use of invertase, an enzyme that can be extracted from bakers yeast. The main objective of
the experiment was to determine the effects of pH in the activity of the enzyme. It involved three parts: (1) the
extraction of invertase, (2) assay of sucrose, and (3) knowing the effects of pH on invertase activity. A chemical
reaction involved was the hydrolysis of sucrose into simpler sugars, glucose and fructose, to react with the DNS
reagent. A red-brown discoloration was observed in the reduction of DNS. A hydrolyzed-sucrose standard curve was
then plotted using the Dinitrosalicylic Method in which the slope of the equation was obtained. Using six different pH
of buffer solutions, the effect of pH was observed on invertase activity. Results show that the optimum pH of the
enzyme invertase was at pH 5.00, where a bell-shaped graph was observed. This means that at this pH, the enzymes
work is at its best.

INTRODUCTION hydrolyze glycosidic (or acetal) linkages with a

Enzymes play an important role in the life
of living organisms. They are biomolecules that
speed up or catalyze chemical reactions. They
maintain and regulate almost all processes in the
body. They can actually be utilized without being
used up in the chemical reaction. Like all
catalysts, enzymes work by providing an
alternative pathway that requires lower activation
energy for a reaction than would otherwise be
required. [1]
substrate specificity for sucrose. The yeast form
of the enzyme has been assigned the unique four
digit enzyme classification code (EC) number of
3.2.1.26 and it is also commonly called -
fructofuranosidase or sucrase. [4]
To purify a specific protein from a mixture
that contains many other proteins, it is essential
to be able to selectively identify and measure the
amount of the target protein without interference
from others in the sample. [4]

Almost all enzymes are proteins which are
folded into complex shapes that allow smaller
molecules to fit into them. The place where these
substrate molecules fit is called the active site. If
the shape of the enzyme changes, its active site
may no longer work because the enzyme has
been denatured. [2]
Different factors can affect enzymatic
reaction. These will include enzyme and substrate
concentration, temperature, and pH. Each factor
highly affects the enzymes shape, structure and
activity.
Changes in pH alter an enzymes shape.
Different enzymes work best at different pH
values. The optimum pH for an enzyme depends
on where it normally works. [2] Below or above
the optimum pH, there is a decrease or increase
in enzymatic activity. Ionic or electrostatic force
is being disturbed once there is a change in pH in
the reaction.
Invertase is an enzyme that catalyzes the
hydrolysis of table sugar (i.e. sucrose) into a
much sweeter, equimolar mixture of glucose and
fructose called invert sugar. [4] Its systematic
name is sucrose glycosidase implying that it is
a member of the subclass of enzymes that
Because of this, the use of Sucrose Assay
must be done using Dinitrosalicylic colorimetric
method must be done for the purpose of
comparison. This method tests for the presence
of free carbonyl group (C=O), the so-called
reducing sugars. It involves the oxidation of the
aldehyde functional group present in, for
example, glucose and the ketone functional
group in fructose. Meaning, 3,5-dinitrosalicylic
acid (DNS) is reduced to 3-amino-5-nitrosalicylic
acid under alkaline conditions. [5]
Invertase splits the disaccharide sucrose
into the monosaccharides glucose and fructose.
Invertase is inhibited by high concentrations of its
substrate, sucrose. The invertase enzyme has
optimum activity at 60 C. Its optimum pH is 4.5
although it is active between pH 3.0 and 5.5.
Inactivation of the enzyme begins at 65 C while
the enzyme is totally inactivated after 5 minutes
at 90 C. [3]
The experiment involves two objectives:
(1) To extract invertase from Bakers yeast and
(2) To determine the effects of changes in pH and
temperature on reaction rates of an enzyme
catalyzed reaction. However, this report will focus
only on the effects of changes in pH on reaction
rates, and not on the effect of temperature.
EXPERIMENTAL
A. Compounds Tested (or Samples used)
This experiment can be divided into three
parts, each using different sets of materials and
reagents. (1) The extraction of invertase from
yeast involved the use of bakers yeast and
distilled water.
(2) For the Sucrose Assay, the following
materials and reagents were used:
conc. HCl
Sucrose solution, 10g/L
Distilled water
0.5M KOH
0.1M buffer solution pH 5.00
DNS reagent
(3) For the effect of pH on Invertase activity,
the following materials and reagents were used:
Invertase stock solution
DNS reagent
DNS reagent
Sucrose solution, 10g/L
0.1M buffer solution pH 1.00
0.1M buffer solution pH 3.00
0.1M buffer solution pH 5.00
0.1M buffer solution pH 7.00
0.1M buffer solution pH 8.00
0.1M buffer solution pH 12.00
B. Procedure
1. The Extraction of Invertase from Yeast
To extract invertase, 0.25g of bakers yeast
was weighed and dissolved into 250mL solution.
From there, the solution was made to stand for
20minutes at room temperature. The supernatant
was collected through decantation and this will
serve as the enzyme stock solution.
100mL of enzyme stock solution was
subjected into boiling water bath for 10 minutes.
This solution will serve as the denatured enzyme
stock solution.
2. Sucrose Assay using Dinitrosalicylic
Colorimetric Method
Seven test tubes were prepared. Table 1
shows the corresponding volume in how the mL
sucrose solution and distilled water were
distributed accordingly.
Table 1. Test Tubes used for the Sucrose Assay
Test Tube mL Sucrose mL distilled
No. standard Solution water
Blank 0.00 1.50
1 0.25 1.25
2 0.50 1.00
3 0.75 0.75
4 0.50 1.00
5 0.25 1.25
6 0.00 1.50
After preparation, three drops of conc. HCl
was added and was subjected to boiling water
bath for 5minutes. Then, 0.15mL of 0.5M KOH
and 2.80mL of 0.1M buffer solution pH 5.00 was
added and mixed thoroughly. Three mL of DNS
reagent was added after and subjected again into
boiling water bath for 10minutes. From here, the
absorbance was measured using the
spectrophotometer. A standard curve was later
constructed.
2. Effect of pH on Invertase Activity
Six test tubes were prepared, each containing
2.90mL of 0.1M buffer solution. Table 2 shows
the pH used for each test tube.
Table 2. pH of 0.1M buffer solutions used
Test 1 2 3 4 5 6
Tube
pH 1.00 3.00 5.00 7.00 8.00 12.0
used 0
After preparation, 0.10mL of enzyme
stock solution was added to each test tube and
was incubated at 60 C water bath for 5minutes.
Then, 1.50mL of sucrose solution was added and
again, incubated at 60 C water bath for
5minutes. Three mL of DNS reagent was then
added and immersed in 95 C water bath for
10minutes to develop the red-brown color. The
solutions were cooled and the absorbance was
measured at 540nm. The hydrolyzed-sucrose
standard curve was used to determine the
amount of sucrose hydrolyzed.
RESULTS AND DISCUSSION
Unlike other carbohydrates, sucrose is the
only non-reducing common disaccharide. Most
tests for sugar detection utilizing such reagents
as Benedict's solution, Fehling's solution, and
DNS (3,5-dinitrosalicylic acid) solution result in
negative readings for sucrose. However, these
methods can still be applied if sucrose is first
hydrolyzed in an acid solution to yield glucose
and fructose. This method is a modification of the
original DNS method for glucose analysis. [6]
 Figure 1 shows the hydrolysis of sucrose The amount of acid hydrolyzed sucrose in
using water to split the bond between the two each test tube can be calculated by getting the
sugar units. concentration of the enzyme stock solution,
which can be done by dividing 0.25g of yeast by
250mL. Meaning it has a concentration of
1mg/1mL. Using the amount of mL, one can
compute for the amount of mg used then divide it
by 4.50mL, the summation of all volume of
mixtures (namely added 0.15mL 0.5M KOH,
Figure 1. Hydrolysis of Sucrose 2.80mL 0.1M buffer, 0.05mL conc. HCl, and the
1.50mL sucrose solution) placed in the solution.
The Dinitrosalicylic Colorimetric Method is
a method that tests for the presence of a free
carbonyl group, which are the so-called reducing Hydrolyzed-sucrose Standard Curve
sugars. It involves the principle of oxidation of
0.8
the aldehyde functional group present in, for 0.59
0.6 0.52
example, glucose and the ketone functional 0.46
0.41 0.41
group in fructose. Meaning, 3,5-dinitrosalicylic f(x) =0.34
1.13x + 0.21
0.4
acid (DNS) is reduced to 3-amino-5-nitrosalicylic Absorbance (540nm) R = 0.61
acid, as shown in Figure 2. [5] 0.20.05
0
0 0.1 0.2 0.3 0.4
Concentration, mg/mL

Graph 1. Hydrolyzed-sucrose Standard Curve

Figure 2. Reduction of DNS
Spectrophotometry is a method to
measure how much a chemical substance
absorbs light by measuring the intensity of light
as a beam of light passes through sample
solution. The basic principle is that each
compound absorbs or transmits light over a
certain range of wavelength. This measurement
can also be used to measure the amount of a
known chemical substance. [7]
The graph was not able to show the direct
proportionality of the amount of sucrose present
to the absorbance due to some inevitable errors.
Using the Sucrose standard curve, it can
determine other concentrations by using the
slope of the equation generated from the given
information (as shown in Graph 1). This is
important as Table 4 now shows the effect of pH
in the absorbance of the solution.

Based from the results of the experiment, Table 4. Effect in changes of pH results
all solutions showed a red-brown discoloration, pH Amount of Acid- Absorbance at
only the blank solution remained yellow (the Hydrolyzed Sucrose 540nm
color of the DNS reagent). This is because it does (mg/mL)
not contain a reducing sugar to react with the 1.00 0.223 0.459 A
DNS reagent to form the 3-amino-5-nitrosalicylic 3.00 0.101 0.321 A
acid. 5.00 0.446 0.710 A
7.00 0.115 0.337 A
Table 3. Results gathered from the DNS Assay 8.00 0.182 0.412 A
Test Tube Amount of Acid- Absorbance at 12.00 0.205 0.438 A
No. Hydrolyzed Sucrose 540nm
(mg/mL) To compute for the amount of acid-
Blank 0 0.052 A hydrolyzed sucrose, you can use the equation
1 0.056 0.456 A generated from the linear regression of the
2 0.111 0.407 A results gathered from the DNS assay. For each
3 0.167 0.342 A pH involved, you can use the slope of the
4 0.222 0.407 A equation to solve any concentration where:
5 0.278 0.520 A
6 0.333 0.585 A y= absorbance at 540 nm and
x = concentration, mg/mL
Graph 2 shows the plotted results of the
effects in the changes of pH.
Effect on changes in pH
0.60.45
0.4
0.22
0.21
0.18
0.20.12
0.1
Amount of Acid-Hydrolyzed Sucrose (mg/mL)
0 Retrieved from:
10
020
pH
Graph 2. Effect on the changes in pH
Based from the graph, it shows that the
optimum pH of the enzyme invertase is at pH
5.00, meaning the activity of the enzyme is at its
peak around this pH.
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. [8]
In conclusion, in a chemical reaction, the
activity of an enzyme is greatly affected by pH. It
can change the shape of the substrate, or even
vary the charges as it contains different acidic
and basic groups since they are amphoteric
molecules. The stability and solubility can also be
affected as there will be no reaction when the
enzyme and substrate cannot identify each other
in the reaction.
REFERENCES
[1] Barron, J. (2014). Enzymes Defined and How
to Buy Them. The Baseline of Health Education
[2] Enzymes:
http://www.bbc.co.uk/schools/gcsebitesize/scienc
e/add_aqa_pre_2011/enzymes/enzymes1.shtml
[3] Invertase by Kerry Bioscience Bioinvert.
http://www.ncbe.reading.ac.uk/ncbe/materials/e
nzymes/invertase.html
[4] Timerman, A. The Isolation of Invertase from
Bakers Yeast An Introduction to Protein
Purification Strategies. Retrieved from:
http://cdn.intechopen.com/pdfs-wm/26595.pdf
[5] Wang, N.S. Experiment 4A: Glucose Assay:
By Dinitrosalicylic Colorimetric Method. Retrieved
from:
http://www.eng.umd.edu/~nsw/ench485/lab4a.h
tm
[6] Wang, N.S. Experiment 9D: Sucrose Assay:
By Dinitrosalicylic Colorimetric Method. Retrieved
from:
http://www.eng.umd.edu/~nsw/ench485/lab9d.h
tm
[7] Spectrophotometry. Retrieved from:
http://chemwiki.ucdavis.edu/Core/Physical_Chem
istry/Kinetics/Reaction_Rates/Experimental_Dete
rmination_of_Kinetcs/Spectrophotometry
[8] Introduction to Enzymes. Retrieved from:
http://www.worthington-
biochem.com/introbiochem/effectsph.html