Enzymes: Effects of pH and Temperature on Invertase Activity

Dayao, Adrian Mark, De Guzman, Ramuel Lance, Dolor, Yoree Arsen,

Ebora, Keziah, Echano, John Patrick
Group 3 2G Pharmacy
Biochemistry Laboratory

ABSTRACT

Enzymes are catalytic protein molecules that speed up the rate of chemical reactions. Enzyme
activity varies due to the different factors such as temperature and pH. In this experiment, the
effect of pH and temperature on the invertase activity were investigated and observed. With the
use of the UV-Vis Spectrophotometer, the absorbance observed was shown to be negative.

INTRODUCTION In Figure 1.0, It shows that increase

Living organisms are made up of
complex yet coordinated structures of
chemical reactions. Now these chemical
reactions are speed up by a chemical called
Catalysts. Catalysts appear in the reaction
mechanism; however, it doesnt appear in
the overall chemical reaction. They can take
many forms such as porous solids and pro-
tein molecules.
Enzymes are catalytic protein mole-
cules with three properties. First, they break
down molecules known as substrate to pro-
duce products but they are not changed or
used up in the process. Second, they in-
crease the rate of chemical reactions. And
lastly, they lower the activation energy for
reactions. Factors, such as temperature,
substrate concentration, and pH, affect the
enzyme activity.
in temperature can denature enzymes thus
they can no longer bind to a substrate. En-
zyme activity will increase until the satura-
tion point is reached where in all enzyme
molecules will bind to substrates. The ob-
jective for this experiment is to investigate
the factors that affect the invertase activity.
METHODOLOGY
Materials
The materials used for this experi-
ment are bakers yeast, test tubes, pipettes,
beakers, marbles, hot plate, ice water, UV-
Vis spectrophotometer. The solutions used
for this experiment are 0.1 M NaHCO3, 0.01
M invert sugar solution, 0.05 M acetate buf-
fer, pH 5.0, 1% dinitrosalicylic acid (DNS)
reagent, and 0.1 M buffer solutions (pH
2,3,5,7,8,12).
Effect of pH on Invertase Activity

First, Test tubes were prepared for

each pH assigned to the groups. Next, 0.50
mL of the 0.1 M buffer solution were added
to each test tube with their appropriate la-
bels. Then, 0.10 mL of the enzyme solution
were added as well as 1.4 mL of distilled
water to each test tube. These test tubes
were further mixed and incubated for 5 min-
utes with the temperature of 60C water
! bath.
Figure 1.0 Factors of Enzyme Activity
 Next, 1 mL of the 0.03 M sucrose RESULTS AND DISCUSSION
were added then incubated once again for 5
minutes in a 60C water bath. Effect of pH on Invertase Activity

After heating, 2 mL of the DNS

reagent were added followed by the immer-
sion of the test tube in a boiling water bath
for 10 minutes to develop a red-brown color
characteristic.

Next, the test tubes were cooled down in an

ice water bath. Lastly, 5 mL of the distilled
water were added to each test tube for dilu-
tion. The absorbance was then measured at
540 nm.

Effect of Temperature on Invertase Activ-

ity

First, a test tube was prepared for

each given temperature water baths which
are 20C, 30C, 40C, 50C, 60C, 70C,
and 90C. 1 mL of 0.03 M sucrose, 1.4 mL
of distilled water, and 0.50 mL of the 0.05 M Figure 2.0 Characteristic of red-brown
acetate buffer solution with a pH of 5.0 were color; given pH = 5
added to each test tubes. These test tubes
were further incubated separately in each Effect of pH
water baths for 5 minutes. 4

Next, 0.1 mL of the enzyme solution 3

was added to the test tubes then further in-
cubated for another 5 minutes without re- 2
moving the test tubes from their respective
water baths. Next, 2 mL of the DNS reagent 1
were added to each test tubes before being absorbance
immersed in a 95C water bath for 10 min- 0
utes which results to developing a red- 2 3 5 7 7.5 8 10 12
brown color characteristic.
Figure 2.1 Plot Graph of Effect of pH
The test tubes were then cooled
down in an ice water bath. Lastly, 5 mL of In Figure 2.1, In the plot graphs
distilled water were added to each test tube
data, it is inferred that the absorbance result
for dilution and then mixed well. The ab-
sorbance was then measured at 540 nm. is negative due to not being able to obtain
the expected bell-shaped curve orientation.
Factors such as inaccurate measurements
due to failure of calibrating laboratory
equipments and personal errors may have
contributed for the negative result in the ex-
periment.

The plot graph also showed the op-

timum pH achieved which is 12. The opti-
mum pH is where the Invertase enzyme is
the most active and it varies for each en-
zyme. In addition, High or low pH results in
alteration of the enzymes shape and loss of
enzymatic activity thus using pH values that
is too high or too low is not highly suggest-
ed.
Figure 2.2 Optimum pH graph
(bell-shaped curve orientation)
Effect of Temperature on Invertase Activ-
ity
Figure 3.0 Characteristic of red-brown color;
given temperature = 50C
Effect of Temperature
3.4
2.55
1.7
0.85
0
25 30 50 70 90 100 100
Figure 3.1 Plot Graph of Effect of Temperature
In Figure 3.1, The plot graph shows
that when the temperature increases the
activity of the enzyme also increases. It is
also shown that there is no drastic change
in the rate of reaction; therefore, the plot
graph did not result into a bell-shaped curve
orientation. Because of it, It is inferred that
the enzyme did not reach denaturation or
that the absorbance result is also negative.
The optimum temperature achieved
is 100C and it is where the enzyme is most
active. Exposure of enzymes to high tem-
perature can cause denaturation.
Figure 3.2 Optimum Temperature graph
(bell-shaped curve orientation)
CONCLUSION
Factors such as pH and temperature
affects enzyme activity wherein high or low
pH and temperature can result in alteration
of the enzymes shape and can cause de-
naturation thus using pH values and tem-
perature that is not on the proper range is
not highly suggested. If an enzyme is dena-
tured, enzymes substrate complexes would
not be able to form thus enzymes would not
be able to catalyse a reaction.

REFERENCES

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