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Effect of pH on Invertase Activity

Kyra Jane N. Wahab
Department of Biochemistry, Faculty of Pharmacy, University of Santo Tomas

ABSTRACT

Enzymes are macromolecular biological catalysts that speed up chemical reactions. This
experiment involves around an enzyme called invertase, which comes from yeast. The objective
of the experiment is to observe the effect of pH on invertase activity and to record its optimum
pH. Four groups were assigned for this task, with each one implementing the same procedures
to their designated pH. The enzyme was first heated in a water bath, then sucrose was added
and was heated again, followed by the addition of the DNS (dinitrosalicylic acid) reagent, which
developed a red brown color after also being submitted to a water bath. The absorbance values
were then achieved through the use of a UV-Vis spectrophotometer. The incorrect optimum pH
of 8.0 was acquired from the data with human error most likely as the suspect.

INTRODUCTION The pH value, wherein the enzyme is most

active, is called the optimum pH. This value
Invertase is extensively scattered
varies from each and every enzyme. [2]
among the biosphere, but it is mainly found
in microorganisms and plants. Bakers yeast This experiment aims to find out the
is the primary strain used for the effects of pH on invertase and find its
commercial production of invertase. [1] optimum pH in which this enzyme works
best.
This enzyme is usually used to
catalyse the hydrolysis of sucrose which is METHODOLOGY
composed of fructose and glucose
Four groups were assigned for this
connected by a glycosidic bond. An equal
activity. The first one, with the pH of 2, the
amount of these two are produced when
second one, 3, the third, 5, and the fourth
this bond is cleaved by the hydrolysis
group, worked with pH 8.
reaction. Invertase is an important enzyme
for photosynthesis. Other than that, it is also Firstly, 0.50 mL of a 0.1 M buffer
used in the confectionary industry. solution with the pH needed by the groups
was added in their respective test tube.
An enzymes effectiveness is greatly
Each test tube was then added with 0.10 mL
affected by temperature and pH. Too big a
of the invertase solution and 1.4 mL distilled
value of either, which causes denaturation,
water. They were mixed thoroughly before
can result in the loss of activity in an
being placed in a 60 oC water bath for five
enzyme while extremely low values
minutes. Afterwards, 1 mL of a 0.03 M
generally result in loss of activity as well.
sucrose was added and the test tubes were
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incubated again in the same 60 oC water Figure 1. Reduction of the DNS reagent
bath for another five minutes. Subsequently,
The absorbance was picked up by
2 mL of DNS reagent was added, and the
the UV-Vis spectrophotometer. The more
test tubes were immersed for the last time in
products that bound with the DNS, the
a boiling water bath under ten minutes. The
higher the absorbance; hence, the
test tubes were covered with marbles in
concentration of products is directly related
order to prevent the mixture from
with the absorbance. The absorbance
evaporating. Lastly, the solutions were
values are found below with 8 as the
pipetted in a microwell plate and the
experimental optimum pH because it had
absorbance values were achieved through
the highest absorbance.
the use of a UV-Vis spectrophotometer.
Table 1. Absorbance values with their
RESULTS AND DISCUSSION
respective pH
The role of the invertase enzyme pH Absorbance
involved the catalyzation of the hydrolysis of 2 0.073
sucrose to its products, glucose and 3 0.103
fructose, which bound with the DNS 5 0.125
[3]
reagent. 8 0.133

The DNS reagent acted as a

This produced a graph, found below,
chromogen, the one that gave the color,
that moved upwards.
red-brown. Originally yellow, the solution
turned red-brown due to the presence of the
free carbonyl group, the so-called reducing
sugar. This involved the oxidation of the
functional groups, aldehyde and ketone,
found in glucose and fructose respectively.
This caused the reduction of the DNS
reagent. [4]

Figure 2. Absorbance graph of the data

Invertase has a relatively high

activity over a broad range of pH from 3.5-
[3]
5.5, with the optimum near the pH 4.5 .
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This implies that the experimental optimum It can be assumed that the
pH was incorrect, as well as the graph inaccuracies of the data are most likely due
created from the data, which was supposed to human errors.
to produce a bell shaped curve.
REFERENCES

[1] Kulshrestha, S., Tyagi P., Sindhi, V.,

Yadavilli, K.S. (2013). Invertase and its
Applications A Brief Review. Journal of
Pharmacy Research, 7(9), 792-797.

[2] The Effect of pH on Invertase Activity.

(n.d). Retrieved from
http://www.markedbyteachers.com/as-and-
a-level/science/the-effect-of-ph-on-
Figure 3. Reference graph invertase-activity.html

The experimental absorbance at pH

[3] Experiment no. 14 Enzyme Kinetics of
8 should have experienced a dip in value
Invertase via Initial Rate Determination.
because a pH too distant from the optimum
(n.d). Retrieved from
number will cause the denaturation of the
https://terpconnect.umd.edu/~nsw/enich485/
enzyme.
lab14.htm
CONCLUSION
[4] Wang, N.S. (n.d). Experiment no. 4A
Although not accurately displayed by Glucose Assay by Dinitrosalicylic
the results, pH proved to be an important Colorimetric Method. Retrieved from
factor in the invertases activity. https://www.eng.umd.edu/~nsw/ench485/lab
4a.htm
Any changes in pH, even slight
ones, would mean that the reaction rate [5] The effect of pH on enzyme activity.
would fall because pH affects the shape of (n.d). Retrieved from
the enzyme as well as the shape or charge http://academic.brooklyn.cuny.edu/biology/bi
properties of the substrate. [5]
o4fv/page/ph_and_.htm