EFFECT OF TEMPERATURE ON INVERTASE ACTIVITY

Hiroaki T.A. Gaviola, Jan D. Gayamo, Claris G. Guiyab,

Sean K. Herman, and Edward O. Jaquiaca
Group 4 2D-Medical Technology General Biochemistry Laboratory

ABSTRACT
Temperature is one of the factors that affects enzyme activity at which the rate of the reaction increases to peak activity
at a certain point and decreases after the said point. The aim of the experiment is to determine the activity of the
enzyme, invertase, when subjected to the following temperatures: 25°C, 50°C, 60°C, and 90°C. Five test tubes were
prepared in which four contained 1 mL of the 0.03 M sucrose, 1.4 mL of distilled water, and 0.50 mL of the 0.05 M
acetate buffer solution with pH 4.7 which were made to react with 0.1 mL of the enzyme solution, 2mL of the
DNS(dinitrosalicylic acid) reagent, and 5 mL of distilled water by the end of the experiment. A blank test tube was also
prepared containing the substrate, water, and buffer. Three drops of each test tube were applied on corresponding wells
on a microwell plate and tested for absorbance at 540 nm using a spectrophotometer. The results for the absorbance
of the blank test tubes for 25°C, 50°C, 60°C, and 90°C were 0.064, 0.224, 0.269, and 0.371 respectively. While the
results for the absorbance of the test tubes with the enzymes were 0.057, 0.376, 0.441, and 0.424.

INTRODUCTION
Enzymes are biomolecules that are made up of An enzyme-catalyzed reaction is known to be
proteins and are present in our body. Enzymes affected by 4 different factors such as the
work by speeding up catalysis of chemical concentration of the enzyme, concentration of the
reactions up to a million times faster. And the substrate, temperature, and pH of the overall
chemical reactions in our body that keeps us alive solution. Generally, enzyme activity increases
and functional depends on the work of enzymes. when enzyme concentration increases, substrate
There are different types of enzymes in the body concentration decreases, and optimal
that can broadly be classified metabolic, digestive, temperature/pH is attained which would usually be
and food enzymes [4]. These different enzymes at a high temperature and an acidic pH but for this
for bodily maintenance function by breaking down experiment, temperature will be the only factor in
large and complex molecules into small and easier focus.
digested molecules, unwinding the double-helix
structure of DNA for replication, and breaking Invertase also known as sucrase or saccharase
down toxins in the body to name a few [3]. is an enzyme extracted from Baker’s yeast and is
the enzyme of discussion in this experiment. It
The mechanism of enzymes are best functions by catalyzing the hydrolysis of sucrose,
represented by the induced-fit model seen in your basic table sugar, into fructose and glucose
Figure _. This shows that when a substrate is usually in the form of inverted sugar syrup which
attracted to an enzyme, the substrate then fits is 1.3 times sweeter than sucrose [2].
onto the active site of the enzyme like a lock fits
to a key. And in order to accommodate the shape
of the substrate more if compatible, the active site
adjusts its shape in order to complement that of
the substrate before breaking it down into a Figure 2. Balanced equation of hydrolysis of
product [3]. sucrose

In contrary to other enzymes, invertase exhibits

a relatively high maximum enzyme activity of
55°C at a given pH of 4.5 and then denatured
when exceeding the said temperature. With this,
the aim of the experiment would then be to
determine the points of which the enzyme activity
of invertase increases and decreases and
determine at which approximate temperature it
exemplifies maximum enzyme activity.

Figure 1. Enzyme and substrate interaction

 added afterwards. The test tubes were immersed
in a boiling water bath for 10 minutes, with the
opening of the tubes covered with marbles to
prevent the solvent from evaporating. This
resulted to the development of a red-brown color.
The test tubes were cooled in an ice water bath,
and were then diluted more with the addition of 5
mL of distilled water. After mixing, the
absorbances of the solutions were measured at
540 nm.

The invert sugar standard curve was made by

plotting the concentration of the invert sugar
Figure 3. Graph showing the influence of
against their corrected absorbances at 540 nm.
temperature on invertase activity
The respective concentrations of each solution was
EXPERIMENTAL obtained by multiplying the invert sugar’s
A. Samples used concentration standard, which is 1.8 mg/mL, to
the volume of the invert sugar solution in each test
Baker’s yeast, 0.1 M NaHCO3, 0.01 M invert sugar tube (refer to the equation below).
solution, distilled water, 1% dinitrosalicylic acid 𝑚𝑔
(DNS) reagent, 0.05 M acetate buffer solution 1.8 𝑥 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑖𝑛𝑣𝑒𝑟𝑡 𝑠𝑢𝑔𝑎𝑟
𝑥= 𝑚𝐿
10
B. Procedure
1. Extraction of invertase from yeast
The corrected absorbances of each solution were
After 10.0 g of baker’s yeast was weighed, it was obtained by subtracting the value of the
dissolved in 30 mL of the 0.1 M NaHCO3. The absorbance of the blank solution from each
solution was left to stand for 24 hours at room solution’s absorbance.
temperature. The supernatant fluid was then
decanted and diluted with distilled water with the 3. Effect of temperature on invertase
ratio being 1:100. The diluted supernatant was activity
used as the enzyme solution.
25°C, 50°C, 60°C, and 90°C water baths were
2. Reducing sugars assay using the prepared. Five test tubes were prepared with each
dinitrosalicyclic (DNS) colorimetric method tube containing 1 mL of the 0.03 M sucrose, 1.4
mL of distilled water and 0.50 mL of the 0.05 M
A series of six test tube were prepared with acetate buffer solution with a pH of 4.7.
different concentrations of invertase sugar diluted
with distilled water. A blank was also prepared in Each test tube was incubated for 5 minutes
another test tube. according to the set water baths with different
temperatures taking note of covering all the test
Table 1. The amount of invert sugar standard tubes with marbles in order to prevent the solution
solution and distilled water in each test tube from evaporating. 0.1 mL of the enzyme solution
was then added into four of the test tubes. One of
Test Tube Invert Sugar Distilled
the test tubes was not mixed with the enzyme
Standard Water (mL)
solution as it will serve as the blank.
Solution (mL)
Blank 0 2.50 The test tubes were then incubated for another
1 0.10 2.40 5 minutes. 2 mL of the DNS reagent was then
2 0.30 2.20 added into each test tube in order to stop the
3 0.50 2.00 reaction to be able to provide data for how much
4 0.80 1.70 enzyme activity took place. The test tubes were
5 1.00 1.50 then immersed in a 95°C water bath for 10
6 1.20 1.30 minutes to develop a characteristic red-brown
color. The test tubes were then cooled by letting
flowing water run through the body of the test
In each test tube, 0.50 mL of the 0.05 M
acetate buffer solution, with pH 4.7, were added
and mixed. 2 mL of the DNS reagent were also
tubes. After cooling, 5 mL of distilled water was 5 0.180 0.164
added to each tube and mixed using a vortex. 6 0.218 0.194

As observed in Table 2, the higher the absorbance

of the sample, the higher the concentration of the
invert sugar. We can then infer that the
concentration of invert sugars is directly related to
the absorbance of the samples.

Figure 4. Solution mixed using a vortex

Each pair of tubes were then subjected to their Figure 5. Invert sugar standard curve
corresponding temperatures of RT, 50°C, 60°C, The standard curve in Figure 5 was made in
and 90°C were applied on the microwell plate by 3 Microsoft Excel by plotting the concentration of the
drops for each well. The absorbance of each tube invert sugar samples against the corrected
was then analyzed and measured by the absorbances of the samples.
spectrophotometer at 540 nm.
2. Effect of Temperature on Invertase Activity
A standard curve was then graphed by plotting
the amount of invert sugar produced against the The rate of enzyme activity in a solution is affected
temperature of the solution. by the solution’s temperature. Each enzyme has
their respective optimum temperature. An enzyme’s
RESULTS AND DISCUSSION optimum temperature is the level of temperature in
a solution at which its rate of activity or reaction is
1. Reducing Sugars Assay Using the
at its highest. However, when an enzyme’s rate of
Dinitrosalicylic (DNS) Colorimetric Method
activity is plotted against the temperature of the
Invert sugar is derived from the hydrolysis of solution, a bell-shaped curve will be seen. This
sucrose and consists of glucose and fructose. The means that when the temperature of a solution
invert sugars are both reducing sugars but sucrose exceeds the optimum temperature of the enzyme,
is a non-reducing disaccharide and does not react in the rate of enzyme activity will decrease.
the carbonyl reactions which are used to determined
The purpose of the DNS was to stop the reaction
reducing sugar capability which is why the DNS
so that the amount of enzyme activity could be
method tests for the presence of free carbonyl
limited and noted for each reaction with a certain
groups and reacts mainly with glucose.
temperature [1]. The DNS reacted with the glucose,
The group was not assigned to this experiment and and the solution with DNS would change color
is using data from the 2nd group since they were the depending on how much sucrose was separated into
ones assigned to do the experiment. glucose and fructose. The more enzyme activity, the
darker the color, and the darker the color the more
Table 2. Corrected absorbances of the standards light would be absorbed by the test tube while in the
and their computed concentration spectrophotometer [1].
Standards Concentration Corrected
of the Invert Absorbances
Sugar
Blank 0.000 0.054
1 0.018 0.017
2 0.054 0.037
3 0.090 0.074
4 0.144 0.075
 Effect of Temperature on Enzyme
Activity
0.25 0.207241
0.183407
0.2
0.15
0.071048
Figure 6. Blank and catalyzed samples applied on a 0.1
microwell plate 0.05 0.002951
0
The following results of absorbance of the blank
0 20 40 60 80 100
and catalyzed tubes were measured by the
spectrophotometer.
Figure 7. Standard curve on the effect of
Table 3. Absorbances of the blank, test tubes with temperature on enzyme activity
the enzyme solution, and the computed corrected
absorbance value The results show that as the temperature
increases, the invertase activity or reaction rate
Blank Absorbance Corrected also increases but only up to a certain point which
Value (with Absorbance is at 60°C and will start to decrease in higher
enzyme sol.) Value temperatures. The decrease in the activity of the
25°C 0.064 0.057 -0.007 enzyme invertase can be attributed to the
50°C 0.224 0.376 0.092 denaturation of its shape. Heat can be used to
60°C 0.269 0.442 0.173 disrupt hydrogen bonds and non-polar
90°C 0.371 0.424 0.053 hydrophobic interactions. This occurs because
heat increases the kinetic energy and causes the
molecules to vibrate violently that the bonds are
The standard curve of the first experiment, disrupted. This then results to the change of the
which is the reducing sugars assay using the DNS shape of the enzyme, making the binding of the
colorimetric method, was used to compute for the substrates to the active site difficult.
amount of invert sugar produced in each test tube.

Table 3. Amount of invert sugar produced and

corrected absorbance value REFERENCES
From the internet (on-line)
Temperature Amount of Corrected
[1] (n.a.), (2017). Effect of temperature on i-
Invert Sugar Absorbance
nvertase activity. Retrieved from
Produced Value
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25°C 0.002951 -0.007 temperature-enzyme-activity/
50°C 0.183407 0.152 [2] Krishna, M., (2009). Why is sucrase called
60°C 0.207241 0.173 invertase? Retrieved from
90°C 0.071048 0.053 http://eamcetzoology.blogspot.com/2009/07/wh
y-is-sucrase-called-invertase.html?m=1
[3] Newman, T., (2018). Enzymes: how they
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