THE INFLUENCE OF PH ON

ENZYMATIC REACTION

Group A17
 Team Members

W Kusuma Naufal Secha Sasha Aprilia

Annisaa A. Ikbar Y. Amelia Rochmat

022011133054 022011133055 022011133056 022011133057

 SUBTOPICS

Tools and Materials

Results

Discussion

Conclusion
 Tools Material
● Test tube and test ● Buffer solution (pH 5)
tube rack ● Substrat Solution 1 %
● Erlenmeyer flask ● NaCl solution 0,9 %
● Spectrophotometer ● HCl solution 0,05 N
● Pipette ● KI-KIO3 solution
● Enzim solution (Amylase)
 0’ 5’ 10’ 15’ 20’

Fill the erlenmeyer flask with

Prepare 5 test tubes and fill each tube with 10 ml 0.05
- 15 ml pH 5 buffer solution
N HCl solution. Mark all tubes with 0’, 5’, 10’, 15’, 20’
- 3 ml substrate solution
- 6 ml NaCl solution

5
 0’ 5’ 10’ 15’ 20’

Add 1 ml of the Erlenmeyer’s solution and put it in a 0’ marked test tube using a pipette

6
 Enzyme solution

0’ 5’ 10’ 15’ 20’

Put 1 ml of enzyme solution into Every 5 minutes, put 1 ml of Erlenmeyer’s solution in

the Erlenmeyer flask. Mix quickly a 5’, 10’, 15’, 20’ marked tube, respectively and mix.
and start the stopwatch.

7
 Shake → wait 5-10 minutes

1 Ml Sol. KI – KIO3 into each tube Read using a spectrophotometer (620

nm)
 The Erlenmeyer tube is filled with
- 15 ml of pH 5 buffer
- solution 3 ml of
- 0.9% NaCl solution 6 ml
Mix the contents of the tube:
Prepare 5 test tubes, mark 0', 5', 10', 15' and 20'. Fill the test tube with 10 ml
of 0.05 N HCl solution.
Add 1 ml of the Erlenmeyer solution and put it in a test tube marked with 0'
using a pipette. Then put 1 ml of the enzyme solution into the Erlenmeyer.
Mix quickly and run the stopwatch.
Every 5 minutes, put 1 ml of the Erlenmeyer solution into the 5', 10', 15' and
20' tubes.
After everything is done, add 1 ml of the KI-KIO3 solution into each test tube,
mix and wait 5 – 10 minutes
Read using a spectrophotometer (620 nm)
Practicum
Results

11
 COLOR :

0’ → dark blue
5’ → pale blue
10’ → yellowish black
15’ → dark yellow
20’ → light yellow
GROUP EXPERIMENT RESULTS TABLE (% DIGESTED SUBSTRATE)

pH 0’ 5’ 10’ 15’ 20’

pH 4 0% 3% 6% 2% 1%

pH 5 0% 12,2% 16,75% 21,15% 27,98%

pH 6,5 0% 64,04% 86,39% 90,11% 91,12%

pH 8 0% 41,83% 69,83% 84,08% 89,5%

pH 10 0% 12,7% 8,7% 8,7% 8,7%

ΔS = percentage of digested substrate

The Effect of pH on Enzyme Activity
TOTAL OF SUBSTRAT IS DIGESTED EACH TIME CAN BE CALCULATED BY

Absorbance t
% substrate is digested = 100% - X 100%
Absorbance t0

Absorbance t0 = absorbance 0’ marked tube

➔ The graph above illustrates that each enzyme has a different optimum pH, making pH
6.5 the most optimum pH for the enzyme, allowing it to function normally at pH 4.

➔ The enzyme's activity dropped dramatically between pH 4 and 10, owing to the enzyme's
acidic and basic pH. The enzyme will be denatured, which will disrupt the enzyme's
active site.

➔ If pH 6.5 is the most optimum, it signifies that enzyme’s nature, activity, and function are
all normal and stable at that pH.
➢pH 4
• The practicum results did not conform to the theory because, at an acid pH (4), the enzyme would
decrease its working activity. It happened because the enzyme was denatured. After all, the protein
structure was damaged. At pH 4 there is an error in the practicum because the digested percentage
decreased.

➢ pH 5
• Amylase enzymes did not work optimally or maximally, but there was a constant increase in the
percentage of digested substrates every 5 minutes.

➢ pH 6,5
• The maximum enzyme performance at the 20th minute. It also showed that the enzyme amylase
worked most optimally at a pH of 6.5 to show a percentage of 91.12%.
➢ pH 8
• Amylase enzymes can work well at a base pH, but the speed of hydrolysis reactions
was slower than the speed of hydrolysis reactions at the optimum pH of amylase
enzymes.

➢ pH 10
• In this reaction, when viewed from 10th, 15th, and 20th-minute data, the amylase
enzyme could not work in a pH 10 atmosphere, so the percentage of hydrolyzed
substrates is small and constant. At pH 10 there is an error in the practicum because
the digested percentage decreased.
Discussion

19
The difference color in each tube indicates the substrate digested by the amylase
enzyme. Amilum is polysaccharide that turns blue when it reacts with iodine. The
amylase enzyme digests amilum in stages, producing a colorless disaccharide, one of
which is a disaccharide when reacted with iodine. The color change that occurs of the
reduction of the blue color indicates that some of the substrate has been digested by the
amylase enzyme. That color change can be measured by a spechtrophotometer.
 Therefore, the enzyme has an optimum pH pH influences the conformation
where the enzyme’s activity is at the highest. structure of an enzyme or substrate.

Enzymes are proteins called The optimum pH of the

biocatalysts. Protein will enzyme, in general, is 5-9
denature if the pH is too low (a bell-shaped curve)
or too high. except pepsin.
 Denaturation
if pH is too low or too high → denaturation
↓
protein structures are damaged except primary structure

primary structure have peptide bonds (strong bond)

 The influence of pH on enzyme’s and
substrate’s charge

Example: pH affect the dimensional structure of the enzyme

E- and SHᐩ → ESH

If pH >
SH+ → S + Hᐩ E = Enzyme
S fails to bind E- SH = Subtrate
If pH <
E- will react with Hᐩ → EH
EH cannot bind SHᐩ
 CONCLUSION
● The optimum pH for enzymes to work
is 6.5.

● The enzym will denature if the pH is

too acidic or too alkaline.

● When the solution in the test tube

clearer, it means the digested substrate
is larger, that means the amount of the
spectrophometry is getting smaller.

● At pH 4 and 10 there is an error in the

practicum because the digested
percentage decreased.
EXERCISE
 1. The working principle of the spectrophotometer

The working principle of the spectrophotometer is the principle of light dispersion which adheres to Lambert Beer's
law. In this law, if monochromatic light passes through one medium, some of the other light will be absorbed and
some will be reflected. While some will be emitted.

Light dispersion is a condition in which white light breaks down into a spectrum of colors. To bring up the dispersion
of light is usually used a prism mirror.

The polychromatic light source then enters the monochromator (this is where the light scatters). In the
monochromator, a spectrum beam is obtained.

From the monochromator then out to the sample cell, in this sample cell there is a process of light absorption by
substances in the sample cell (where the incoming light is brighter than the light after leaving).

The light is then captured by the detector and converted into an electric current.
 2. The function of HCl, NaCl, KI-KIO3, Buffer Solution
and Enzyme Solution :
HCl serves to create an acidic atmosphere so that iodine is released from KI-KIO3 (potassium iodate) and iodine is available
to bind to non-hydrolyzed substrates. Iodine with a substrate that is not hydrolyzed will form a color that can be an
indicator of how much of the substrate is hydrolyzed. HCl also serves to stop the work of enzymes.

Cl from NaCl acts as an enzyme activator.

KI-KIO3 serves as a color indicator for spectrophotometer readings. When reacting with HCl Iodine is released and react
with amilum.

Buffer solution serves to provide pH for experiment and maintain the pH during the experiment

Amylase enzyme as a catalyst. Amylase plays a role in breaking down starch molecules through the hydrolysis process
3. What is the initial velocity of an enzymatic reaction,
and how is it measured?
Initial velocity is the velocity at which the curve is just progressing, with the substrate content
still not decreasing. The initial speed of the reaction can be determined by measuring the
decrease in substrate content or increase in product content during the reaction. The way to
measure the initial velocity is by calculating it with a Progress Curve.
4. Factors that can affect the activity of an enzyme

a. pH
b. Temperature
c. Enzyme rate
d. Substrate rate
e. Modifier : Inhibitor dan Aktivator
THANK YOU!