Extraction of Invertase, Glucose Assay and Effects of pH on the Reaction Rates of an

Enzyme-Catalyzed Reaction
Christine Mae E. Reyno, Jericho Luis E. Rivera, Theresa Marice P. Rivera, Marjorie Anne A.
Roo, Chelsea Ann G. Rubio, Khristine Grace A. Salamat
Group 9
2C Medical Technology
Biochemistry Laboratory
ABSTRACT
Invertase was extracted from Bakers yeast. Through Dinitrosalicylic Colorimetric Method, the glucose standard curve
was plotted on which the amount of acid-hydrolyzed sucrose was based. Using a sucrose solution, the effects of
changes in pH on the reaction rates of an enzyme-catalyzed reaction were determined. Sucrose, a non-reducing
sugar, was reduced into its simpler sugars-fructose and glucose to react with DNS to give a red-brown discolorizaton
of which its absorbance was measured. The results showed a bell-shaped graph in which the optimum pH to hydrolyze
sucrose is identified at approximately 5.5. The experiment showed that at certain pH and temperature, enzymes are
denatured thereby losing their function.

INTRODUCTION
At any given moment, all of the work being
done inside any cell is being done by enzymes.
Enzymes are biological catalysts - catalysts are
substances that increase the rate of chemical
reactions without being used up. [1]
Most enzymes are proteins that 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. We say the enzyme has been
denatured. They can be denatured by high
temperatures or extremes of pH. [2]
The activity of enzymes is strongly affected by
changes in pH and temperature. Each enzyme
works best at a certain pH and temperature, its
activity decreasing at values above and below
that point. This is not surprising considering the
importance of (1) tertiary structure (i.e. shape)
in enzyme function and (2) noncovalent forces,
e.g., ionic interactions and hydrogen bonds, in
determining that shape [2]
Sucrose, commonly known as table sugar, is a
disaccharide composed of an alpha-D-glucose
molecule and a beta-D-fructose molecule linked
by an alpha-1,4-glycosidic bond. When this bond
is cleaved in a hydrolysis reaction, an equimolar
mixture of glucose and fructose is generated.
This mixture of monosaccharides is called invert
sugar, which is derived from the fact that sucrose
rotates plane polarized light to the right i.e.,
dextrorotatory, +66.5, whereas the hydrolysis
products rotates plane polarized light to the left

i.e., levorotatory, -20 for the mixture (+52.5

for D(+)-glucose and -92 for D(-)-fructose). [3]
Invertase (maxinvert L 1000, systematic
name: beta-fructofuranosidase) is an enzyme
that catalyzes the hydrolysis (breakdown) of
sucrose (table sugar). [4] Its systematic name,
beta-fructofuranosidase, implies that the reaction
catalysed by this enzyme is the hydrolysis of the
terminal
non-reducing
beta-fructofuranoside
residues in beta-fructofuranosides. Sucrose can
be hydrolyzed relatively easily; the reaction
proceeds in an acidic environment without the aid
of invertase. [4]
Maxinvert is obtained from bakers yeast,
Saccharomyces cerevisiae. Invertase splits the
disaccharide sucrose into the monosaccharides
glucose and fructose shown in the equation:
Sucrose +
glucose + fructose
Invertase is inhibited by high concentrations of
its substrate, sucrose. The invertase 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. [5]
Invertase is mainly used in the food
(confectionery) industry where fructose is
preferred over sucrose because it is sweeter and
does not crystallize as easily. However, the use
of invertase is rather limited because another
enzyme, glucose isomerase, can be used to
convert glucose to fructose more inexpensively.
For health and taste reasons, its use in food
industry requires that invertase be highly
purified. [5]

This experiment has two goals: (1) To extract

invertase from Bakers yeast and (2) To
determine the effects of changes in pH and
temperature on reaction rates of an enzymecatalyzed reaction.

EXPERIMENTAL
A. Compounds tested (or Samples
used)
1.For extraction of Invertase from Yeast
Bakers yeast
Distilled water
2. For Sucrose Assay
0.5M KOH
0.1M Buffer solution

DNS reagent

Glucose Standard Solution

3. For Effect of pH on Invertase Activity
3.87 M Buffer Solution
4.0 M Buffer Solution
5.5 M Buffer Solution
7.3 M Buffer Solution
10.55 M Buffer Solution
Invertase stock solution
DNS reagent
Sucrose Solution
B. Procedure
1. Extraction of Invertase from
Yeast
0.25g of Bakers yeast was weighed
into an Erlenmeyer flask using a triple
beam
balance.
Afterwards,
it
was
dissolved in distilled water to make a
250ml solution. The solution was allowed
to stand for 20 minutes at room
temperature.
Through
filtration,
the
supernatant was collected and was
labelled as the invertase stock solution.

2. Glucose Assay using

Dinitrosalicylic Colorimetric
Method
Seven (7) well-labelled test tubes were
prepared with the following volumes of
solutions added:
Test
(ml) mL Glucose
Tube Standard
No.
Solution
Blank
0
1
0.25
2
0.5
3
0.75

(ml) mL Distilled
Water
1.50
1.25
1.00
0.75

1.00

0.50

1.25

0.25

1.50

Table 1. Series of test tubes for Glucose

Assay
0.15 ml of KOH was then added to each test
tube to neutralize the solution. Next, 2.80 ml of
0.1 M buffer solution of pH 5 was added into the
test tubes and were mixed well. Subsequently, 3
ml of DNS reagent was put into each solution and
were immersed into 95 C water bath for 10
minutes to develop the characteristic red-brown
color. After cooling, the absorbance was
measured using the spectrophotometer. The
Glucose Standard Curve was later constructed.

3. Effect of pH on Invertase Activity

Six numbered test tubes were prepared and
were added 2.90ml of appropriate buffer
solutions as described below.
Tube
no.

pH of
Buffer
soln

3.87

4.0

5.5

7.3

10.55

Table 2. Effect of pH buffer solutions

0.10 ml of invertase stock solution was added
to each test tube, mixed thoroughly and all test
tubes were incubated in 60C water bath for 5
minutes. 1.50 ml of sucrose solution was added
and the reaction mixture was incubated in 60C
water bath for 5 minutes. Then, 3 ml of DNS
reagent was added into each test tube and were
immersed in 95C water bath for 10 minutes to
develop the characteristic red-brown color..
After cooling, the absorbance was measured at
540nm. The blank test tube which was used on
the glucose assay was retained and was used as
the blank to autozero the spectrophotometer at
this part of the experiment. The amount of
sucrose hydrolysed was determined using

the hydrolysed-sucrose standard curve.

RESULTS AND DISCUSSION
The Dinitrosalicylic Colorimetric Method tests

for the presence of free carbonyl group

(C=O), the so-called reducing sugars. This
involves the oxidation of the aldehyde

functional group present in glucose and the

ketone
functional
group
in
fructose.
Simultaneously,
3,5-dinitrosalicylic
acid
(DNS) is reduced to 3-amino,5-nitrosalicylic
acid under alkaline conditions:

Figure 3. Actual Dinitrosalicylic Colorimetric

Method Results

Figure 1. Reduction of DNS

The above reaction scheme shows that one
mole of glucose will react with one mole of 3,5dinitrosalicylic acid.

Figure 2. Reduction of Sucrose

Sucrose is a non-reducing sugar and therefore
does
not
undergo
reaction
with
3,
5
dinitrosalicylic acid. Thus, sucrose must be
broken down into simple sugars. DNS, a normally
yellow solution, when added into solutions
containing reducing sugars (e.g glucose) change
in color to red-brown due to the reduction of the
DNS reagent into 3-amino-5-nitrosalicylic acid.
Glucose and other reducing sugars react with
DNS by oxidation reaction producing a colored
compound that shows a maximal molar extinction
at 540nm. Glucose is reduced into gluconic acid.
The more glucose present in the solution, the
more intense will the discolorization be.
In the experiment, only the blank solution
didnt showed a red-brown discolorization and
stayed as a yellow solution, the color of DNS
reagent, because it lacks a reducing sugar that
will react with DNS reagent to form 3-amino-5nitrosalicylic acid.

A spectrophotometer can measure the amount

of light absorbed by a solution, and the quantity
of light absorbed is proportional to the intensity
of color or pigment in the solution. A second
principle involved in spectrophotometry is that
the amount of light absorbed is
also dependent on through how much of the
solution
the
light
must
pass.
The
spectrophotometer measures absorbance. [6]
Absorbance values, by themselves, do not
describe the concentration of a substance.
However, we can determine the concentration of
a substance in a solution using a standard curve.
A standard curve translates absorbance values
into concentration. Using the spectrophotometer,
we do not directly measure the concentration of
glucose. Instead, we measured the absorbance of
colored product created when glucose reacts with
the Glucose Assay Reagent. The amount of
colored substance is directly proportional to the
amount of glucose present.
Test Tube No.

Blank
1
2
3
4
5
6

Amount
of
AcidHydrolyzed
Glucose
(mg/ml)
0.56
1.12
1.67
2.22
2.78
3.33

0.000 A
2.414 A
2.67 A
2.74 A
2.82 A
2.84 A
2.87 A

Table 3. Dinitrosalicylic Colorimetric Method

Results
The concentration of glucose in each tube was
calculated using the following equation:
V1C1= V2C2
Where
V1 = volume of the stock solution
C1 = the concentration of the stock solution

V2 = the final diluted volume (volume of stock

solution and diluent)
C2= the final diluted concentration

Test tubes with pHs 4.0, 5.5, 7.3, and 10.55

follow the same procedure in solving for the
amount of acid-hydrolyzed sucrose.

Effect of pH

The result will be divided by the distribution

factor, 4.5 to give a result of 0.56. The same
principle was used in computing for the
concentration of glucose in the other test tubes.

Absorbance (540 nm)

Glucose Standard Curve

y = 1.1611x
R = -0.049

2
0
1

Concentration, mg/ml

Figure 4. Glucose Standard Curve

pH
3.87
4.0
5.5
7.3
10.55

Anount of
AcidHydrolyzed
Sucrose
(mg/ml)
0.140
0.153
0.840
0.130
0.127

0.162
0.178
0.975
0.151
0.148

1.5
1
0.5
0
0

10

15

pH

Figure 5. Effect of pH Graph

Absorbance (540 nm)

(100ml)X = (1000mg)(0.25ml)
X = 2.5 mg

A
A
A
A
A

The graph shows a bell-shaped curve wherein

the optimum pH to hydrolyze sucrose is at
approximately 5.5. The most favorable pH value the point where the enzyme is most active - is
known as the optimum pH. The centre (optimum
pH) and breadth of this 'bell-shaped' curve
depend upon the acid dissociation constants of
the relevant groups in the enzyme. [7]
The rates of many enzymatic reactions adhere
to a bell shaped curve when they are a function
of pH. These curves reflect the ionization state of
the amino acid residues that must have a specific
ionization state for enzymatic activity to take
place. The observed pK's (maxima point) often
hints at the identity of the amino acid residues
which are essential for enzymatic activity. [8]

Table 4. Effect on pH Results

CONCLUSION

The
concentration
of
glucose
can
be
determined either by calculating it or using the
graph. Using the standard curve, we can
determine the concentration of other solutions by
locating the absorbance of that solution on the yaxis and drawing a horizontal line to the standard
curve then you can draw a vertical line from that
intersection to the x-axis to determine the
concentration. On the other hand, to calculate it,
divide its absorbance by the slope of the standard
curve as shown below:

The rate of a chemical reaction and/or the

enzyme activity is influenced by the pH. A change
in the pH affects the rate of reaction. When pH of
a particular medium changes, it leads to
alteration in the shape of the enzyme. The pH
level may also affect the charge properties and
shape of the substrate. Within a narrow pH
range, changed in the structural shapes of the
enzymes and substrates may be reversible but

For pH 3.87, the Absorbance is 0.162 A and the

slope of the graph is y=1.1611x.
0.162 = 1.1611x
X=
X= 0.140

for a significant change in pH levels, the

enzyme and the substrate may undergo
denaturation. In such cases, they cannot
identify each other. Consequently, there will
be no reaction as such. pH affects enzyme
activity.

REFERENCES
[1] How cells work by Marshall Brain
http://science.howstuffworks.com/life/cellularmicroscopic/cell2.htm
[2] Enzymes
http://www.bbc.co.uk/schools/gcsebitesize/scien
ce/add_aqa_pre_2011/enzymes/enzymes1.shtml
[3] Enzyme Kinetics of Invertase via initial rate
determination Prepared by Nam Sun Wang
http://www.eng.umd.edu/~nsw/ench485/lab14.h
tm
[4] Invertase
http://en.wikipedia.org/wiki/Invertase
[5]Invertase by Kerrt Bioscience Bioinvert
http://www.ncbe.reading.ac.uk/ncbe/materials/e
nzymes/invertase.html
[6] Spectrophotometric Measurement of Glucose
http://employees.csbsju.edu/mcampos/bio114/la
bmaterials/lab.2.writeup.03.pdf
[7]Enzyme Technology Effect of pH and ionic
strength by Martin Chaplin 26/12/12
http://www1.lsbu.ac.uk/water/enztech/ph.html
[8]Structural Biochemistry/Enzyme/Effects of pH
on enzyme activity
http://en.wikibooks.org/wiki/Structural_Biochemi
stry/Enzyme/Effects_of_pH_on_enzyme_activity