ENZYMES

Kassandra Duran, Jose Encabo, Ryan Enriquez, Marion Agatha Esguerra, Raemee Hanna Facon
Group 5 2B Medical Technology Biochemistry Laboratory
ABSTRACT
Enzymes are biochemical catalysts essential in the human body. These enable the biochemical reactions inside the
human body to occur in a much faster rate without altering the reaction. Because of the structure and composition of
enzymes, changes in pH and temperature affect its activity. In this experiment, the enzyme activity of invertase were
tested on various pH and temperatures. The samples were then tested though the spectrophotometer to measure the
absorbance of each which underwent different pH and temperatures. The results were then used to solve for the best
fit line of the samples tested at various pH and temperature which may be used to derive the pH and temperature at
which the enzyme invertase has its optimal activity.

INTRODUCTION rate, due to denaturation of the protein structure

There are various chemical reactions that enable
the human body to work. These chemical
reactions, such as DNA replication, are sped up or
catalyzed by enzymes. Enzymes can make a
chemical reaction millions of times faster than it
would have been without it. [6]
Enzymes are examples of a catalyst - a
substance that speeds up a chemical reaction—
without being a reactant. Enzymes are catalysts
for biochemical reactions that happen in living
organisms. These are usually proteins, though
some ribonucleic acid (RNA) molecules act as
enzymes. [5]
Enzymes perform the critical task of lowering a
reaction's activation energy—that is, the amount
of energy that must be put in for the reaction to
begin. Enzymes work by binding to reactant
molecules and holding them in such a way that the
chemical bond-breaking and bond-forming
processes take place more readily. Enzymes lower
the energy of the transition state, an unstable
state that products must pass through in order to
become reactants. [7]
Blanco and Blanco stated that enzymes are
catalysts that, within the mild conditions of
temperature, pH, and pressure of the cells, carry
out chemical reactions at amazing high rate. [3].
A general rule of thumb for most chemical
reactions is that a temperature rise of 10°C
approximately doubles the reaction rate. To some
extent, this rule holds for all enzymatic reactions.
After a certain point, however, an increase in
temperature causes a decrease in the reaction
rate, due to denaturation of the protein structure
and disruption of the active site. On the other
hand, a general rule of thumb for most chemical
reactions is that a temperature rise of 10°C
approximately doubles the reaction rate. To some
extent, this rule holds for all enzymatic reactions.
After a certain point, however, an increase in
temperature causes a decrease in the reaction
and disruption of the active site. [1]
In this experiment, the objective is to determine
the effects of changes in pH and temperature on
the reaction rates of enzyme-catalyzed reactions.
EXPERIMENTAL
A. Test Compound/s or Sample/s
used
The samples used were invertase, sucrose,
dinitrosalicyclic acid (DNS) reagent and acetate
buffer solution.
B. Procedure
Effect of Room Temperature on
Invertase Activity
Six test tubes were prepared each of which
contained 1 mL of 0.03 M sucrose, 1.4 mL of
distilled water and 0.50 mL of 0.05 M acetate
buffer solution (pH 4.7). The test tubes were left
for 5 minutes. Then, 0.1 mL of the invertase was
added to the six test tubes. Again, the test tubes
were left for 5 minutes. 2 mL of DNS reagent was
added to the six test tubes and the color of the
solution became a characteristic red-brown. 5 mL
of distilled water was added to each of the six test
tubes. Afterwards, the test tubes were mixed
vigorously. Another test tube was prepared
containing 1 mL of 0.03 M sucrose, 1.4 mL of
distilled water and 0.50 mL of 0.05 M acetate
buffer solution (pH 4.7). 2 mL of DNS reagent was
added to the test tube and the color of the solution
became a characteristic red-brown. 5 mL of
distilled water was added to the test tube.
Afterwards, the test tube was mixed vigorously.
Three drops of each of the prepared solutions were
put into a microwell. The microwell was then
subject to the spectrophotometer which measured
the absorbance of the solutions at 540 nm.
RESULTS AND DISCUSSIONS 3 7.5 -0.006 -0.0265
Table 1. Sucrose Assay using the Dinitrosalicylic
Colorimetric Method 4 12 -0.014 -0.0606

Amount Invert Absorbance

Sugar Produced
(mol/L) Solutions for concentration of sample solutions:

Blank:
Blank 0 0 1:

−0.024 = 0.2344𝑥 + 2.1485 × 10−4

Standard 1 0.018 3.0 x 10-3
0.2344𝑥 = −0.024 − 2.1485 × 10−4
Standard 2 0.054 0.015
0.2344𝑥 −0.02421485
=
0.2344 0.2344
Standard 3 0.090 0.020
𝑥 = −0.1033

Standard 4 0.126 0.032 2:

−0.021 = 0.2344𝑥 + 2.1485 × 10−4

Standard 5 0.180 0.041
0.2344𝑥 = −0.021 − 2.1485 × 10−4
0.2344𝑥 −0.02121485
=
r2 = 0.988 0.2344 0.2344
𝑥 = −0.0910
Straight Line Equation: y=0.2344x+2.1485 x 10-4

Graph 1. Standard Curve

3:
Standard Curve −0.006 = 0.2344𝑥 + 2.1485 × 10−4
0.05
y = 0.2332x + 0.0004 0.2344𝑥 = −0.006 − 2.1485 × 10−4
ABSORBANCE

0.04
R² = 0.9825
0.03 0.2344𝑥 −0.00621485
=
0.02 0.2344 0.2344
0.01 𝑥 = −0.0265
0
0 0.05 0.1 0.15 0.2 4:
CONCENTRATION −0.014 = 0.2344𝑥 + 2.1485 × 10−4

0.2344𝑥 = −0.014 − 2.1485 × 10−4

Table 2. Effect of pH on Invertase Activity 0.2344𝑥 −0.0142
=
0.2344 0.2344
pH Absorbance Amount
Invert 𝑥 = −0.0606
Sugar
Produced In Table 2, it showed the different absorption
(mol/L) measurement of the invertase when it was
exposed to different pH levels. The absorbance
was measured using the spectrophotometer When
1 2 -0.024 -0.1033 it was exposed to pH 2 the absorbance was -0.024,
at pH 4.9 it was -0.021, at pH 7.5 it was -0.006,
2 4.9 -0.021 -0.0910 and at pH 12 it was -0.014. This showed that the
pH level which the absorbance activity was at the
peak was at pH 7.5, however, the data gathered
Blank 22°C 0 -9.17x10-4
was incorrect. In a research, the optimum pH for
invertase is at pH 4.5 (average) [4]. The reason
was that invertase operated best at pH 4.5, and 5 22°C -0.004 -0.0180
when the pH level increased, the conformation of
invertase was altered, thus ceased the substrate
to fit perfectly in the active site [2]. Table 2 also 6 60°C 0.010 0.0417
showed the amount of invert sugar produced. For
pH 2 the amount produced was -0.1033 mol/L, for 7 70°C 0.006 0.0247
pH 4.9 it was -0.0910 mol/L, for pH 7.5 -0.0265
mol/L and for pH 12 it was -0.0606 mol/L. The
amount of invert sugar produced was solved with 8 80°C 0.052 0.2209
the equation of the line y = 0.2344x + 2.1485 x
10-4. Solutions for concentration of sample solutions:

Graph 2. Effect of pH on Invertase Activity Blank:

0 = 0.2344𝑥 + 2.1485 × 10−4
pH VS Amount of Invertase 0.2344𝑥 −2.1485 × 10−4
=
Sugar 0.2344 0.2344

0 𝑥 = −9.17 × 10−4
Amount of Invert Sugar

0 5 10 15
5:
-0.05
−0.004 = 0.2344𝑥 + 2.1485 × 10−4

-0.1 0.2344𝑥 = −0.004 − 2.1485 × 10−4

0.2344𝑥 −0.00421485
-0.15 =
pH 0.2344 0.2344
𝑥 = −0.0180
In graph 2, it showed the relationship between the 6:
pH level and the invertase activity. The graph 0.010 = 0.2344𝑥 + 2.1485 × 10−4
should have shown a bell-shaped standard curve.
The curved started low because the enzyme did 0.2344𝑥 = 0.010 − 2.1485 × 10−4
not work well in low pH or acidic environments. 0.2344𝑥 0.00978515
The curve gradually started to rise which meant =
0.2344 0.2344
that the invertase activity also rose. When the
curve reached the peak (4.5 pH), the invertase 𝑥 = 0.0417
activity was at its highest, where it became most 7:
active and reached the optimum pH level. As the 0.006 = 0.2344𝑥 + 2.1485 × 10−4
temperature rose, the invertase activity gradually
declined until it went back to the lowest invertase 0.2344𝑥 = 0.006 − 2.1485 × 10−4
activity. This meant that after the optimum pH 0.2344𝑥 0.00578515
level, the invertase became denatured thus made 0.2344
=
0.2344
it hard for the substrate to bind with the enzyme
at the active point. 𝑥 = 0.0247

Table 3. Effect of Temperature on Invertase 8:

Activity 0.052 = 0.2344𝑥 + 2.1485 × 10−4

0.2344𝑥 = 0.052 − 2.1485 × 10−4

Temperatu Absorbanc Amount
re e Invert 0.2344𝑥 0.005178515
=
Sugar 0.2344 0.2344
Produced
𝑥 = 0.2209
(mol/L)
In table 3, it can be seen that invertase, when it
was exposed to different temperatures, showed
different absorption activity. The absorbance was
measured with the spectrophotometer. At room
temperature (22𝆩C) the absorbance was -0.004,
at 60°C, it was at 0.010, at 70°C it was 0.006, and
at 80°C it was 0.052. This meant that at 80°C, the
invertase showed the greatest rate of reaction.
However, the data was incorrect. In a research, it
was said that the optimum temperature that
showed the greatest reaction was at 55-60𝆩C [4].
The reason why these were the optimum
temperature was that because, just like for pH, the
conformation of the enzyme did not yet change
when it reached these temperatures. When the
temperature went beyond 60°C, the conformation
changed and ceased the substrate to attach to the
active site of the enzyme [2]. It can also be
observed that the amount of invert sugar
produced at room temperature (22°C) was -
0.0180 mol/L, at 60°C it was 0.0417, at 70°C it
was 0.0247 and at 80°C it was 0.2209. The
amount was calculated with the equation of the
line y=0.2344x+2.1485 x 10-4.
Graph 3. Effect of Temperature on Invertase
Activity
Temperature VS Amount of
Invertase Sugar
0.3
Amount of Invert Sugar
0.2
0.1
0 from
0 20 40 60
-0.1
Temperature (C)
In graph 3, it showed the relationship between the
temperature and the invertase activity. The graph
should have shown a bell-shaped standard curve.
The curved started low because the enzyme did
not work well in low temperatures. The curve
gradually started to rise which meant that the
invertase activity also rose. When the curve
reached the peak (60°C), the invertase activity
was at its highest, where it became most active
and reached the optimum temperature. As the
temperature rose, the invertase activity gradually
declined until it went back to the lowest invertase
activity. This meant that after the optimum
temperature, the invertase became denatured
thus made it hard for the substrate to bind with
the enzyme at the active point.
REFERENCES
[1] Ball, D., et al. (n/a). The Basics of General,
Organic, and Biological Chemistry. LibreText
Libraries. Retrieved from
https://chem.libretexts.org/Bookshelves/Int
roductory_Chemistry/Book%3A_The_Basics
_of_GOB_Chemistry_(Ball_et_al.) on
October 15, 2019.
[2] Bettelheim, F. A., Brown, W. H., Campbell, M.
K., Farrell, S. O., & Torres, O. (2012).
Introduction to general, organic and
biochemistry. Nelson Education.
[3] Blanco, A. and Blanco G. (2017). Medical
Biochemistry. Academic Press. Retrieved
from
https://www.sciencedirect.com/topics/neur
oscience/enzymes on October 15, 2019.
[4] Kaur, N. K., Kaur, M. K., Gupta, A. K., & Singh,
R. K. (1992). Properties of β‐fructosidases
(invertases and inulinases) of Fusarium
oxysporum grown on an aqueous extract of
Cichorium intybus roots. Chemical
Technology and Biotechnology. Retrieved
80 100 https://onlinelibrary.wiley.com/doi/abs/10.
1002/jctb.280530308 on October 15, 2019
[5] Khan Academy. (n/a.) Enzymes and the active
site. Retrieved from
https://www.khanacademy.org/science/biol
ogy/energy-and-enzymes/introduction-to-
enzymes/a/enzymes-and-the-active-site on
October 15, 2019.
[6] Newman, Tim. (2018). Enzymes: how they
work and what they do. Medical News Today.
Retrieved from
https://www.medicalnewstoday.com/article
s/319704.php on October 15, 2019.
[7] OpenStax, Biology. OpenStax CNX. May 14,
2015 Retrieved from
http://cnx.org/contents/185cbf87-c72e-
48f5-b51e-f14f21b5eabd@9.85 on October
15, 2019.