ENZYMES

Extraction of Invertase from Yeast, Dextrose-Fructose Assay Using Dinitrosalicylic Colorimetric

Method, Effect of pH and Effect of Temperature on Invertase Activity

#1 Abinsay, Katrina Maria, #2 Adalid, Alvin John, #3 Alexander, Maureen Theresa,

#4 Almazan, Jan Aira, #5 Ambagan, Eldrick Justin
2E-PH
ABSTRACT
Extraction of invertase from Bakers Yeast and to determine the effects of changes in pH
and temperature on reaction rates of an enzyme-catalyzed reaction were the objectives of this
experiment. Dinitrosalicylic Colorimetric Method was used to determine the concentration of the
sucrose (dextrose & fructose) and the absorbance values were measured with a UV-Vis
Spectrophotometer. Results show the conversion of 3,5-dinitrosalicylic acid to 3-amino-5nitrosalicylic acid with the formation of red-brown coloration from yellow. Glucose was also
converted to gluconic acid. The pH on invertase activity gave a bell-shaped curve and the pH
was accurate to the expected result, ph 5, which is within a small range of pH. And the
temperature showed imperfect bell-shaped because of the initial optimum value of 20C, which
is considered inconclusive, which was then replaced by the 2nd highest peak, 70C, also
considered inaccurate given the standards for the expected result which is of body temperature
(37.5C).

INTRODUCTION
In every living cell, even the
simplest organisms, have hundreds of
enzymes which all are part of catalyzing
reactions that are crucial in life [1]. Enzymes
increase the chemical reaction rate taking
place within living cells without them
suffering any overall change, from which
they are called, biological catalysts.
Substrates are the reactants of enzymecatalyzed reaction [2]. Enzymes specificity
is highly acknowledged that usually
catalyzes only one type of reaction. Some
show absolute specificity, some show less,
many of which show stereoisomeric
specificities. Generally, enzyme molecules
are significantly considered larger than
substrate molecules with an exception of
proteinases, nucleases and amylases all of
which acts on macromolecular substrates.
The binding site of the substrate is called the
active site, a cleft or pocket in the surface of

the enzyme that constitutes a part of the

enzyme molecule. Factors affecting the
governance of the rate of enzyme-catalyzed
reaction are temperature, pH and
concentration of enzyme and substrate [3].
Sucrose or cane sugar (C12H22O11) is
a white crystalline solid, soluble in water,
sweet in taste and is dextro-rotatory (+66.5).
It is hydrolyzed in equal amounts of glucose
(+) and fructose (-) by the treatment of
dilute acid or sucrase (invertase) that will
change dextro-rotatory to levo-rotatory as a
whole, where the magnitude of levo-rotation
(-92) of fructose is greater than the
magnitude of dextro-rotation (+52.7) of
glucose. This conversion of optical rotation
is termed as Inversion. And the products,
glucose and fructose, are called invert sugars
[4]
.

L-glucose (Levorotatory glucose) is the

other isomer of glucose, which is hardly
found on nature [8].
Figure 1: Sucrose Inversion [5]
Invertase, also named as -fructofuranosidase or sucrase, acts on the
hydrolysis of sucrose giving off glucose and
fructose separately. Specifically, the
inversion is done by the hydrolysis of the
terminal non-reducing beta-fructofuranoside
residues in beta-fructofuranosides [6].

Figure 3: Chemical structure of 3,5dinitrosalicylic acid [9]

Figure 2: Chemical structure of Glucose [7]

Glucose (C6H12O6), also termed as
dextrose, D-glucose, or grape sugar, is a
simple monosaccharide found in plant. It is
part of the three dietary monosaccharide,
together with fructose and galactose, that are
directly absorbed into the bloodstream
during digestion. Used as a primary source
of energy and a metabolic intermediate, a
reason why it is an important carbohydrate
in biology. It is one of the main products of
photosynthesis and also a part of cellular
metabolism. Glucose can be seen in
different types of molecular structure, which
all can be divided into two families of
mirror-images (streoisomers). In nature,
only one set of these isomer exists, which
are the D-glucose (right-handed form of
glucose). Cellulose and starch polymers are
derived from the dehydration of D-glucose.

3,5-dinitrosalicylic
acid
(C7H4N2O7), also known as 2-Hydroxy3,5-dinitrobenzoic
acid,
3,5-Dinitro-2hydroxybenzoic acid, 3,5-Dinitrosalicylate,
Benzoic acid,
and 2-hydroxy-3,5[10]
dinitrosalicylic acid . 3,5-DNS is used in
DNS Colorimetric Method, which tests for
the presence of free carbonyl functional
group (C=O), termed as sugars, where
oxidation of the aldehyde functional group is
also involved. For instance, oxidation of
glucose and the ketone functional group of
fructose seen in Figure 4 and the reduction
of 3,5-DNS to 3-amino-5-nitrosalicylic acid
seen in Figure 5 [11].

Figure 4: Oxidation reaction [12]

Figure 5: Reduction of 3,5-DNS [13]

degradation. This occurs because when the

enzyme is held at high temperature for a
long period of time, where the possibility of
a cooked enzyme may be seen [15]. A perfect
illustration of the effect of temperature of
enzyme reaction is a bell-shaped curve
shown in Figure 6.

Figure 6: Temperature in enzyme reaction

[14]

Figure 7: pH in enzyme reaction [15]

Temperature is one of the factors
affecting enzyme reaction and is basically
needed for a reaction to occur where
collision of molecules with the energy equal
to or greater than the activation energy
happens. Involvement of heat in a reaction
makes it faster to occur which makes it
collide more often so. Approximately, a
10C increase in temperature will double the
rate of reaction. When a reaction reaches its
peak, where enzyme activity is at its fastest
occurrence, is called the optimum
temperature. Generally, enzyme reactions
peak is at body temperature (37.5C), high
increase in such will create an enzymeenzyme reaction variation, where a dramatic
fall in reaction rate is observed. In addition,
when enzymes are heated the proteins chains
extra energy and their mobility is more
possible. Breaking down of proteins will
occur first on weaker bonds, then on Van
Der Waals attraction between side groups
and lastly, their hydrogen bonds [14]. This
fall is termed as denaturation, where the
stability of proteins decreases due to thermal

Enzymes are proteins, all of which

are very sensitive to pH changes. Optimum
range of pH is its peak, where reaction is
most active. Results of the effect of pH vary
on a combination of factors, which are as
follows: the binding of the enzyme to
substrate, the catalytic enzyme of enzymes,
the ionization of the substrate, and the
variation of protein structure [15]. Figure 7
shows the perfect bell-shaped curve, where a
concrete enzyme reaction to pH is seen.
METHODOLOGY
Reagents and Materials:
- Bakers Yeast
- Sucrose standard solution (dextrose +
fructose) 100mg/mL
- Concentrated HCl
- 0.5 M KOH
- Dinitrosalicylic acid (DNS) reagent
- 0.1 M buffer solutions (pH 2, 3, 5, 7, 11)
- Sucrose solution, 10 g/L
- Test tubes
- Pipettes

- Beakers
- Volumetric flask
- Paraffin film
- Hot plate
- UV-Vis spectrophotometer
a. Extraction of Invertase from Yeast
Bakers yeast, weighing 0.25g, was
dissolved in a distilled water to make a 250mL solution. For 20 minutes, the solution
was allowed to stand at room temperature.
The supernatant then was collected (The
supernatant serves as the enzyme stock
solution that will be used for the succeeding
experiments).
b. Preparation of Denatured Invertase
Stock Solution
Enzyme stock solution (100mL) was
incubated in a boiling water bath for 10
minutes and was allowed to cool. The
supernatant then was collected (This serves
as the denatured enzyme stock solution that
will be used for the succeeding
experiments).
c. Sucrose Assay using Dinitrosalicylic
Colorimetric Method
A series of test tubes was prepared,
all of which are labeled as follows:
Test Tube
No.
Blank
1
2
3
4
5
6
7
8

mL Sucrose
Standard
Solution
0
1.15
1.20
1.25
1.30
1.35
1.40
1.45
1.50

mL Distilled
Water
1.50
0.35
0.30
0.25
0.20
0.15
0.10
0.5
0

All tubes that are prepared were covered

with marbles to prevent evaporation of the

solvent.
Three
drops
(~0.05mL)
concentrated HCl was added to each tube
and was mixed well. At 90C, the tubes
were incubated on a water bath for 5
minutes. Next was the addition of 0.15 mL
0.5 M KOH to neutralize the solution.
Another was the addition of 2.80 mL 0.1 M
buffer solution (pH 5) and was mixed well.
DNS reagent (3 mL), was added to the
solution. The test tubes were immersed in
95C water bath for 10 minutes
(Development
of
red-brown
color
characteristic was expected). After cooling,
the absorbance at 540 nM was measured.
Hydrolyzed-sucrose standard curve was
constructed by plotting A540 against
concentration (mg/mL).
d. Effect of pH on Invertase Activity
Five test tubes were prepared which
are labeled as follows:
Test Tube No.
1
2
3
4
5

pH of Buffer solution
(0.1M, 2.90 mL)
2
3
5
7
11

Enzyme stock solution (0.10 mL) was added

to each tube and was individually mixed
thoroughly. All tubes were then incubated in
60C water bath for 5 minutes. Next was the
addition of 1.50 mL of sucrose solution and
the reaction mixture was incubated in 60C
water bath for 5 minutes. DNS reagent was
then added to each with 3 mL measurement.
The test tubes were immersed in 95C water
bath for 10 minutes (Development of redbrown color is expected). Blank solutions
were prepared by following the previous
steps noting that Denatured enzyme was
used instead of enzyme stock solution.
Lastly, the absorbance at 540nm was
measured.

e. Effect of Temperature on Invertase

Activity
Six test tubes were prepared, which
all are labeled as follows:
Test tube
1
2
3
4
5
6

Temperature, C water
bath
20
30
50
60
70
90

The test tubes were given 1.50 mL of

sucrose solution each and were all incubated
separately in 5 minutes on a water bath in
respect their assigned temperature. Then,
0.10 mL of enzyme stock solution was
added to each and 3 mL of dilute enzyme
solution was mixed after (0.1M, 2.90 mL
buffer solution). The test tubes were then
incubated for 5 minutes at their assigned
temperatures. After that, 3 mL of DNS
reagent was added to each tube, all of which
were immersed in a 95C water bath for 10
minutes (Development of red-brown color is
expected). Blank solutions were prepared by
following the previous steps noting that
Denatured enzyme was used instead of
enzyme stock solution. Lastly, the
absorbance at 540nm was measured.
RESULTS AND DISCUSSION
1. Sucrose (Dextrose & Fructose) assay
using
Dinitrosalicylic
Colorimetric
Method
In the experiment, a change in color
occurred, where a transparent solution of
dextrose and fructose (sucrose) with a
yellow solution of DNS, formed a redbrown solution after heating. Glucose being
the reducing agent, inducing a reduction of

3,5-dinitrosalicylic acid forming 3-amino-5nitrosalicylic acid, where it decreased its

number of oxygen and an increase in the
number of hydrogen. On the other hand,
oxidation of glucose to gluconic acid
occurred, where DNS acted as the oxidizing
agent. Both of which noted that Oxidation is
a reversible chemical reaction where one of
the reactions is oxidation and the one with
the reverse reaction is reduction.

Figure 8: DNS and Glucose RedOx reaction

Concentrations were computed as follows:
Test tube blank = _100 mg_ = ___x___
1000 mL
0 mL
X = 0.0 mg
7.5 mL
X = 0.0 mg/mL
Test tube 1 = _100 mg_ = ___x___
1000 mL 1.15 mL
X = 0.115 mg
7.5 mL
X = 0.016 mg/mL
Test tube 2 = _100 mg_ = ___x___
1000 mL 1.20 mL
X = 0.12 mg
7.5 mL
X = 0.0167 mg/mL
Test tube 3 = _100 mg_ = ___x___
1000 mL 1.25mL
X = 0.125 mg
7.5 mL
X = 0.0167 mg/mL
Test tube 4 = _100 mg_ = ___x___

1000 mL 1.30 mL
X = 0.13 mg
7.5 mL
X = 0.0173 mg/mL
Test tube 5 = _100 mg_ = ___x___
1000 mL 1.35 mL
X = 0.135 mg
7.5 mL
X = 0.018 mg/mL
Test tube 6 = _100 mg_ = ___x___
1000 mL 1.40 mL
X = 0.14 mg
7.5 mL
X = 0.0187 mg/mL
Test tube 7 = _100 mg_ = ___x___
1000 mL 1.45 mL
X = 0.145 mg
7.5 mL
X = 0.0193 mg/mL

Test tube 8 = _100 mg_ = ___x___

1000 mL 1.50 mL
X = 0.15 mg
7.5 mL
X = 0.02 mg/mL
Test Tube
No.

Concentration
mg/mL

Blank
1
2
3
4
5
6
7
8

0
0.015
0.016
0.0167
0.0173
0.018
0.0187
0.0193
0.02

Figure 9: Sucrose Assay Best Fit Line

Absorbance
at 540 nm
(A)
0
0.702
0.337
0.578
0.244
0.241
0.192
0.331
0.283

Using
a
spectrophotometer,
absorbance was identified to each
concentration, seen at Figure 9. The best fit
line determines the relationship between
each concentration in accordance to their
absorbance. In the experiment, according to
the trendline measured from Microsoft
excel, only 1 was penetrated having a slopeintercept form of y=14.21x +0.100 and a
linear regression of R2=0.173. This means
that there is minimal relationship between
each point, which may be caused by the
following: Inactivity of sucrose (not freshly
prepared), the DNS might not be reactive,

and the spectrophotometer might not be

sensitive enough to read the measurements.
2. Effect of pH on Invertase Activity
Test Tube
No.

pH

1
2
3
4
5

2
3
5
7
11

Absorbance
at 540 nm
(A)
-0.209
-0.047
0.175
0.021
-0.140

Figure 10: Effect of pH Standard Curve

Figure 10 shows that the ideal shape
was formed, which is the bell-shaped
formation of the plots. The optimum pH is
pH 5 indicating that the enzyme reaction
will be at its most active state. It states that

enzyme only works within a small pH range

this is because, as the pH changes, there will
be an impulsive break of intramolecular and
intermolecular bonds affecting the shape of
the invertase together with its effectiveness.

After pH 5, graph shows a decrease in

absorbance as the pH increases, which notes
a denaturation of the invertase [16].
3. Effect of Temperature on Invertase
Activity

Test Tube
No.

Temperature
(C)

1
2
3
4
5
6

20
30
50
60
70
95

Absorbance
at 540 nm
(A)
1.024
-0.105
-0.121
-0.138
0.980
-0.217

Figure 11: Effect of Temperature Standard Curve

As the temperature increases,
Figure 11 shows that at 20C the
denaturation of the invertase begun, all of
invertase activity is at its optimum reaction
which can be seen at temperatures 30C,
rate, indicating a very low temperature
50C and 60C. As the increase in
compared to the ideal temperature which is
temperature continues, reaching 70C,
Body temperature (37.5C), which in this
absorbance increased again, indicating an
experiment, is considered an inconclusive
increase in invertase activity, being the
result, for it doesnt contribute to the main
second peak of the graph, which will be
invertase activity seen on the bell-shaped
considered as the most active invertase
curve.
activity because of its partaking on the bellshaped curve. After which, a decrease on

absorbance is again seen as the temperature

further increases at 95C indicating a
denaturation of the invertase.
Generally,
the
results
are
inconclusive, for it doesnt show accuracy
on the ideal optimum temperature which
must be near or around the body temperature
(37.5C), from which, results show that the
optimum temperature is 70C that in fact is
very high for an enzyme molecule to gain
more for its kinetic energy. Another reason
is that, at higher temperature or above body
temperature,
intramolecular
and
intermolecular bonds are broken down, so a
denaturation at 95C is too late to occur for
it reaches already a surplus of heat standard
[16].
Negative results are seen on pH and
temperature absorbance, all of which are due
to the following factors: The blank absorbs
more light than the sample (The
luminescence phenomenon cannot give
more light output than the incident radiation
because the number of photons emitted
cannot exceed the number of incident
photons.), and Instrument calibration
(Reference sample for baseline correction is
contaminated moisture or other surface
impurities) [17].
CONCLUSION
In conclusion, invertase extracted
from the Bakers yeast catalyzed the
reaction towards the hydrolysis of sucrose
into glucose and fructose, which were used
as reducing sugars from the Red-Ox reaction
with DNS, forming a red-brown color.
Measurement of the absorbance led to a
non-linearized form of the absorbance given
by each concentration of the assay, giving
off a single penetrated plot causing a
population of outliers, all are defined to be
caused by errors of accuracy and misleading
equipments. The effect of pH and

temperature both were illustrated as bellshaped curve, only to differ on the

temperatures
early
appearance
of
misleading outlier, showing an initial
optimal temperature. On the other hand,
both the bell-shaped curve still gave an
optimum pH and temperature, therefore
concluded respectively, only to note, an
inaccuracy on the expected value for optimal
temperature, which is higher than it was
presumed. Also, both the pH and
temperature exhibited negative absorbance
values, all of which are manifested by
procedural errors and inaccuracy of
laboratory equipments.
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