I.

Tittle : The Influence of pH and Enzyme Concentration to Its Activity

II. Date : Wednesday, March 12th 2018 at 9.30 – 12.00
III. Purpose of Experiment : To prove that both pH and concentration of enzyme influences
its activity
IV. Basic Theory
A. Enzymes
Enzymes are catalysts and increase the speed of a chemical reaction without
themselves undergoing any permanent chemical change. They are neither used up in the
reaction nor do they appear as reaction products.
The basic enzymatic reaction can be represented as follows
S + E → P + E (Bennet, 1969)
where E represents the enzyme catalyzing the reaction, S the substrate, the substance
being changed, and P the product of the reaction.
Enzymes belong to proteins that speeds up the rate of a chemical reaction in a
living organism. Knowledge of basic enzyme kinetic theory is important in enzyme
analysis in order both to understand the basic enzymatic mechanism and to select a
method for enzyme analysis. The conditions selected to measure the activity of an
enzyme would not be the same as those selected to measure the concentration of its
substrate. Several factors affect the rate at which enzymatic reactions proceed -
temperature, pH, enzyme concentration, substrate concentration, and the presence of any
inhibitors or activators. The activity of every enzyme will increase as the temperature
increasing until the optimum temperature is reached (Megiandari, 2009). Increasing
enzyme concentration will increase the rate of reaction, as more enzymes will be
colliding with substrates molecules. However, this too will only have an effect up to a
certain concentration, whree the enzyme concentration is no longer the limiting factor.
Enzymes are affected by changes in pH. The most favorable pH value - the point where
the enzyme is most active - is known as the optimum pH. This is graphically illustrated in
Picture 1.

Picture 1. Effect of pH on reaction rate

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 Enzymes are very specific. Each has a particular job it does and it does only that
job. Digestive enzymes are enzymes that break down food into usable material. The
major different types of digestive enzymes are: amylase, protease, lipase, cellulase, etc.
Enzymes amylase are enzyme which can be act as catalyst in amylum hydrolysis by
wtare form sugar. The processing starch or amylum into sugar by enzyme as catalyst
consist of two steps, which are the liquefaction and the saccharification. In the
liquefaction, 𝛼-amylase (alpha-amylase) helps amylum hydrolysis process to be
oligosachharides. Amylases are starch degrading enzymes. Initially, the term amylase
was used originally to designate enzymes capable of hydrolzing 𝛼-1,4-glucosidic bonds
of amylosa, amylopectin, glycogen and their degradation product (Bernfeld, 1955). Two
categories of amylases, denoted alpha and beta, differ in the way they attack the bonds of
the starch molecules.
Enzyme 𝜶-amylase
Alpha- amylase is widespread among living organisms. In the digestive
systems of humans and many other mammals, an alpha amylase called ptyalin is
produced by the salivary glands. Alpha- amylase is extracelluler enzyme that
could cut 𝛼-1,4-glucosidic linkage between the monomer of glucose in linear
chain of amylose. The meccahnisms of starch hydrolysis by α-amylase is shown
below :

α-amylase

amylum

α-maltose

α-glucose

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 The characteristic and source of α-amylase that produce dextrin (Wahyuni,
2015).
Source enzyme Molecular weight Stable T (oC) Optimum pH
Salliva Human 62000 50 6,8 – 7
Human Pancreas 56000 50 6,8 – 7
Malt 41500 60-66 5,5
Rhizopus 51000 <40 4,6 – 4,8
Strepcocus 79000 45 5,6

Basicly, α-amylase activity testing can be done by three prime observation

which are an increase in the strength reduction, decreasing the intensity of the
blue colour, and changing the density of optical. A decreasing in the intensity of
the blue colour iodin-amylum (starch) complex compound happened because of
the amount of the substrat will diminidh caused by enzyme activity in hydrolyzing
the starch (Yoo, 1987).
B. Starch or Amylum
Starch is carbohydrates found in plants. It consist of two different types of
polysachharides that are made up of glucose units which are connected in two
different ways. One is the linear amylose and the other is the branched amylopectin.
Amylose is the compoud in amylum that is responsible for the blue colour. Its chain
forms a helix shape, and iodine can be bound inside the helix. Molecular iodine (I2)
form polyiodide ions of the type In- when potassium iodide is added. In the case of
the aqueous solutions of polyiodides, the absorptions o the different species lead to an
overall brownish colour. Once amylose is added, it forms another CT (charge-
transfer) complex, here, the amylose acts as a charge donor and the polyiodide as an
acceptor. This complex absorbs light of a different wavelength than polyiodide, and
the colour turns dark blue (Sheri, 2016).
C. The spectronic UV-Vis
Ultraviolet and visible (UV-Vis) absorption spectroscopy is the measurement
of the attenuation of a beam of light after it passes through a sample or after reflection
from a sample surface. Absorption measurements can be at a single wavelength or
over an extended spectral range.
The absorbance of the starch-iodide complex is measued to calculate blue
value and the apparent percentage amylose content can be determined by developing

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 a blue value standard curve with purified amylose. The absorbance usually at 620 nm
- 680 nm.
Below is colors of different wavelenght region (Gary, 2014).
Wavelength Absorbed Transmitted colour
absorbed (nm) colour (complement)
380–450
Violet Yellow-green

450–495
Blue Yellow

495–570 Green Violet

570–590
Yellow Blue

590–620
Orange Green-blue

620–750 Red Blue-green

V. Tools and Materials :

A. Tools
1. Spectronic UV-Vis
2. Water Bath
3. Beaker glass
4. Pippetes
5. Volumetric flask
6. Cylinder flask
B. Materials
1. Salliva 1 ml
2. Aquades Sufficiently
3. Iodine Solution Sufficiently
4. Starch Solution Sufficiently
5. pH 1 Solution Sufficiently
6. pH 3 Solution Sufficiently
7. pH 5 Solution Sufficiently
8. pH 7 Solution Sufficiently
9. pH 9 Solution Sufficiently

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VI. Flow Chart
1. The Influence of pH
a. Preparation of sample

1 ml of salliva

- Diluted with aquades 10x

Result (diluted
salliva)

b. Blanko Solution
1 ml of Starch solution

- Added 0,5 ml of aquades

- Heated in 37oC for 5 minutes
- Added 2 drops of iodium
- Added 6 ml of aquades
- Read the absorbance in wavelength 680 nm

The Absorbance

c. Test Solution

1 ml of 1 ml of 1 ml of 1 ml of 1 ml of
Starch Starch Starch Starch Starch
solution + 1 solution + 1 solution + 1 solution + 1 solution + 1
ml of pH 1 ml of pH 3 ml of pH 5 ml of pH 7 ml of pH 9

- Added 0,5 ml of Salliva

- Heated in 37oC for 5 minutes
- Added 2 drops of iodium
- Added 6 ml of aquades
- Read the absorbance in wavelength
680 nm

The Absorbance

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 2. The Influence of Concentration
a. Preparation of sample
1 ml of salliva

- Diluted with aquades 10x, 20x,

30x, 40x, and 50x

Result (diluted
salliva)

b. Blanko solution

1 ml of Starch solution + 1
ml of PH Optimum

- Added 0,5 ml of aquades

- Heated in 37oC for 5 minutes
- Heated in 70oC for 15 minutes
- Added 2 drops of iodium
- Added 6 ml of aquades
- Read the absorbance in wavelength
680 nm
The Absorbance

c. Test Solution

1 ml of 1 ml of 1 ml of 1 ml of 1 ml of
Starch Starch Starch Starch Starch
solution + 1 solution + 1 solution + 1 solution + 1 solution + 1
ml of pH ml of pH ml of pH ml of pH ml of pH
optimum optimum optimum optimum optimum

- Added 0,5 - Added 0,5 - Added 0,5 - Added 0,5 - Added 0,5
ml saliva ml saliva ml saliva ml saliva ml saliva
10x diluted 20x diluted 30x diluted 40x diluted 50x diluted

- Heated in 37oC for 5 minutes

- Heated in 70oC for 15 minutes
- Added 2 drops of iodium
- Added 6 ml of aquades
- Read the absorbance in wavelength 680 nm

The Absorbance
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VII. Result of Observation

N Result Observation
Procedur Hypothesis / Reaction Conclusion
o Before After
1 a. Preparation of Sample  Salliva =  Salliva + pH
Colourless aquades = Optimum pH of alpha-amylase from optimum of
 Starch colourless human sallive is pH 6.8 – 7 (Wahyuni, alpha
solution = 2015) amylase
Colourless  Starch + aquades a. Hydrolisis based on
 pH (1,3,5,7, = colourless the
and 9)  Starch + aquades experiment
solution = + heated = is pH = 9
colourless colourless
 iodine solution
b. Blanko Solution solution =  Starch + aquades 𝛼-Amylase
yellowish + heated +
brown iodine = dark
blue solution
+ aquades =
light blue b. Amylum-iod complex
solution
The absorbance
= Attached

 Starch solution + + nI2

pH (1,3,5,7, and →
9) = colourless
solution
 Starch solution +
pH (1,3,5,7, and
9) + salliva =
colourless
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 c. Tested Solution solution
 Starch solution +
pH (1,3,5,7, and
9) + salliva +
heated in 37oC =
colourless
solution
 Starch solution +
pH 1 + salliva +
heated in 37oC +
iodine = dark
blue
 Starch solution +
pH 3 + salliva +
heated in 37oC +
iodine = blue
solution (+)
 Starch solution +
pH 5 + salliva +
heated in 37oC +
iodine = blue
solutin (+++)
 Starch solution +
pH 7 + salliva +
heated in 37oC +
iodine = blue
solution (++)
 Starch solution +
pH 9 + salliva +
heated in 37oC +
iodine = blue
solution (++++)
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  + 6 ml aquades
= light blue
solution
 The Absorbance
= attached

2 a. Preparation of Sample  Salliva =  Salliva + From the

Colourless aquades =  Hydrolisis experiment
 Starch colourless we can
solution = conlude that
Colourless  Starch + pH 9 + concentrati
 pH 9 aquades = on of
solution = colourless enzyme
colourles  Starch + pH 9 + influence
 iodine aquades + the activity
solution = heated = . The more
b. Blanko Solution yellowish colourless concentrate
brown solution d the more
 Starch + pH 9 +  Amylum-iod complex its activity.
aquades + In 10 times
heated + iodine diluted,
= dark blue activity of
solution enzyme is
+ aquades = + nI2 most
light blue →
solution
The absorbance CH2OH CH2OH
O O
I
= Attached I
OH H OH
I

O O O
n
 H OH OH I
 Starch solution + I I

pH 9 + salliva
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 (10x, 100x,
1000x, 10000x,
and 100000x) =
colourless
solution
 Starch solution +
pH 9 + salliva
c. Tested Solution (10x, 100x,
1000x, 10000x,
and 100000x) +
heated =
colourless
solution
 Starch solution +
pH 9 + salliva
10x + heated +
iodine =
colourless
 Starch solution +
pH 3 + salliva
100x + heated +
iodine =
colourless
 Starch solution +
pH 5 + salliva
103x diluted +
heated+ iodine =
blue solution (+)
 Starch solution +
pH 7 + salliva
104x diluted +
heated + iodine
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 = blue solution
(++)
 Starch solution +
pH 9 + salliva
105x diluted +
heated + iodine
= blue solution
(+++)
 The Absorbance
= attached

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VIII. Analysis And Explanation
The experiment that already started and done at Wednesday, September 12th 2018 at
9.30 – 12.00 have a title “The Influence of pH and Enzyme Concentration to Its Activity”
and have purpose to prove that either pH or enzyme concentration influence enzyme
activity. The first experiment has pH as manipulation variable. The purpose is to know
which pH optimum is. In pH optimum, the activity of enzyme also will be optimum. The
second experiment has concentration of enzyme as manipulation variable. The purpose is to
know the influences of concentration of enzyme to its activity. In this experiment, we use
iodine to know the existence of amylum both in blanko and tested solution then detect the
activity of enzyme. Besides, it also can be conducted by testing the existence of glucose as
indicator the activity of enzyme.
A. The Influence of pH to its activity
This experiment has goal which is determining which pH that make activity of
enzyme can be maximum. Enzyme amylase is used here. There are three type of
amylase. Alpha amylase, beta amylase and gama amylase. Alpha amylase (also know as
ptyalin) is produced by the salivary glands. We choose salliva as a sample because it is
easy to be taken. Salliva is colourless. The first step to do its experiment is sample
preparation. One mililiter of salliva is diluted with aquades in 10 ml volumetric flask. It
is called 10 times dilution.
The second step, blanko solution is made as a standard of tested solution. It
should receive the same treatment as tested solution but one variable which is
manipulated. One mililiter of starch solution is added 0,5 ml of aquades. Afterthat, it is
mixed well and heated in 37oC for 5 minutes. Then, added 2 drops of iodine which is
yellowish brown into the solution. The colourless solution become dark blue.
The next step is making tested solution. Five test tubes are prepared and labelled
(we give label 1, 3, 5, 7, 9 show the pH). Each tube is filled by 1 ml of starch solution
and added the pH solution. In test tube which is labelled 1, 1 ml of pH 1 is added into
there and so do all. pH solution is colourless. Afterthat, it is mixed well and heated in
37oC for 5 minutes in waterbath. The enzyme work at optimum pH and temperature. The
enzyme will hydrolize the amylum and yield the glucose. If the pH is not in optimum
condition, much amylum will be left and vice versa. Below is the reaction of hydrolysis
amylum by enzyme.

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 α-amylase

amylum

α-glucose

. Then, added 2 drops of iodine into the solution. Although the intensity of colour
is different, there are two tubes which is the colour as same as blanko solution (dark
blue). In blanko solution, dark blue is formed because of the reaction which is occured
between amylum in starch and iodin.
There are two function of iodin addition in tested solution, that are as indicator
how much amylum is left and giving colour to the solution. Below is the reaction

(aq) + nI2 (aq) →

Reddish brown

colourless

(aq)

Dark Blue

After that, all of tube such as blanko and tested solution (1, 3, 5, 7, and 9) is
added 6 ml of aquades. This addition has function to dilute the solution in order to give
the valid data (absorbance) in the spectronic uv-vis because its limit of detection is 1. If
the colour of solution is too concentrated, the spectronic will give the data only based on
13 |
 its assumption. So it is not valid. Then, the solution (blanko and tested solution) is read
the absorbance by 680 nm wavelength.
Enzyme (in this case alpha-amylase) will hydrolize the amylum and yield
glucose. But, it works as its condition. In this experiment, the enzyme is added pH
solution. Theoretically, optimum pH of enzyme alpha amylase in salivary glands of
human is about 6,8-7. If the solution too acid or in the below of optimum pH, the works
of enzyme will be not maximum or optimum. If the solution too base or in the above of
optimum pH, the enzyme will be denaturated. Enzyme belongs to protein, it has four
shape that is quartener, tersier, sekunder and primer. The most complex is quartener
shape and the most simple is primer shape. So, enzyme will be broken to the simple
shape if pH is too base.
The relation between absorbance and activity of enzyme
The absorbance show the concentration of iodine-amylum complex. The result of
absorbance in the experiment is shown in table below.
No Tube Absorbance in 680 nm
1. Blanko Solution 0.156
2. 1 (pH 1) 0.149
3. 3 (pH 3) 0.021
4. 5 (pH 5) 0.035
5. 7 (pH 7) 0.023
6. 9 (pH 9) 0.020

From the table, it can be figured the graph

relation between pH and A
0.16
0.14
0.12
0.1
0.08
Y-Values
A

0.06
Linear (Y-Values)
0.04
0.02
0 y = -0.0128x + 0.1136

-0.02 0 2 4 R² = 0.5245
6 8 10
pH

14 | Graph 1. Relation between pH and A

 Relation between pH and ΔA
0.18
0.16 y = 0.0128x + 0.0424
0.14 R² = 0.5245
0.12 0.135 0.136
0.133
0.1 0.121

ΔA
0.08 Series 1
0.06
Linear (Series 1)
0.04
0.02
0
0 0.007 2 4 6 8 10
pH
Graph 2. Relation between pH and ∆A
The absorbance show how much amylum is left in hydrolisis reaction by the
enzyme. The more amylum, the bigger the absorbance. The more amylum, the lower
activity of enzyme so it can’t hydrolize the amylum into glucose. Based on the graph
realtion between pH and the absorbance, the lowest absrobance is in pH 9. It show that
the amylum which is left is the fewest. While in the graph relation between pH and ∆A,
show the contrast. So, the optimum pH is in pH 9.

B. The Influence of pH to its activity

After the optimum pH is found. The second experiment has purpose the
influences of enzymes concentration to its activity. The manipulation variable here is
concentration of enzyme. Ten times-diluted salliva that we have is diluted again into 100
times, 103 times, 104 times, and 105 times. The way to dilute is taking 1 ml of solution
from ten times dilute, then dilute it in 10 ml volumetric flask and so on– if we want to
make 100 times diluted. So, in this second experiment we have five sample in different
concentration. The same with the first experiment, blanko solution is made as a standard
and has treatment as same as tested solution has. One mililiter of starch is added pH
optimum and so is 0.5 ml of aquades. Afterthat, that solution is heated in 37oC for 5
minutes and in 70oC in 15 minutes relatively. Two drops of iodine is added and 6 ml of
aquades is too.
The next step is making tested solution. Five test tubes are prepared and labelled.
Each tube is filled by 1 ml of starch solution and added the optimum pH solution. In test
tube which is labelled 1, 0.5 ml of salliva 10 times-diluted is added. In test tube which is
labelled 2, 0.5 ml of salliva 100 times-diluted is added, so onwards. Afterthat, it is

15 |
 mixed well and heated in 37oC for 5 minutes in waterbath followed by heated in 70oC
for 15 minutes. The function of the first heating as same as the first heating in the first
experiment which is to speed up the enzyme activity because it has optimum
temperature. Meanwhile, the second heating is to stop the activity of enzyme because if
the temperature is too high, the enzyme will be broken. That high temperature (70 oC) is
the minimal temperature which can break the enzyme. The activity of enzyme should be
stop here because the enzyme is in its optimum pH, so it will works optimally and
hydolize all of the amylum. We need amylum to remain left so it can react with iodine
and can be seen in spectronic. Hereinafter, 2 drops of iodine is added into the solution in
five different test tubes. In the tube 1, the colour of solution remain colourless after
iodine addition. It shows that the remain amylum is such a little. While in the tube 2,3,4,
and 5, the solution is blue with different intensity. It is caused by the amylum which is
left in each tube is different. After that, all of tube such as blanko and tested solution (1,
2, 3, 4, and 5) is added 6 ml of aquades. This addition has function to dilute the solution
in order to give the valid data (absorbance) in the spectronic uv-vis because its limit of
detection is 1. If the colour of solution is too concentrated, the spectronic will give the
data only based on its assumption. So it is not valid. Then, the solution (blanko and
tested solution) is read the absorbance by 680 nm wavelength.
The absorbance show the concentration of iodin-amylum complex. The result of
absorbance in the experiment is shown in table below.

No Tube Absorbance in 680 nm

1. Blanko Solution 0.022
2. 1 (10 x) 0.007
3. 2 (100 x) 0.017
4. 3 (1000 x) 0.020
5. 4 (10000 x) 0.015
6. 5 (100000 x) 0.022

Just like the first experiment, the absorbance show how much the amylum which
is left. So in greatest absorbance, it indicates that the enzyme doesn’t work optimally
because the amount of amylum in the 105 times dilution as same as in blanko solution
which there is not enzyme there. It can be figure in a graph below.

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 Relation between Concentration and A
0.025
y = 7E-08x + 0.0145
0.02 R² = 0.3178 0.022
0.02
0.0150.017
0.015

A
Y-Values
0.01
Linear (Y-Values)
0.0050.007

0
-50000 0 50000 100000 150000
Concentration (the number of dilution)
Graph 3. Relation between concentration and A
From the graph, it can be interpreted that the absorbances have up and down
accordance to the concentration. Based on the theory, increasing enzyme concentration
will increase the rate of reaction, as more enzymes will be colliding with substrates
molecules. So it is should have linear graph from left the bottom to the right on.
However, this too will only have an effect up to a certain concentration, where the
enzyme concentration is no longer the limiting factor.

So, the optimum concentration in this experiment is in 10 times dilution

because it has the lowest absorbance (see in graph 3). We see that between graph 3 and
4 is antipodes.

Relation between Concentration and ΔA

0.015
0.016
0.014
0.012
0.01
0.007
0.008
ΔA

0.005 Y-Values
0.006
0.0040.002 Linear (Y-Values)
0.002 0
y = -7E-08x + 0.0075
0
R² = 0.3178
-20000
-0.002 0 20000 40000 60000 80000 100000 120000
Concentration (the number of dilution)

Graph 4. Relation betweenconcentration and ∆A

17 |
IX. Conclusion
From the explanation above, we can conclude that :
1. The optimum activity of enzyme is on its optimum pH which is pH 9
2. The optimum activity of enzyme is on its 10x dilution (in the fewest concentration)
X. References
Bennett, T. P., and Frieden, E.: Modern Topics in Biochemistry, pg. 43-45, Macmillan,
London (1969).
Bernfeld P. 1955. Amylase, 𝛼 and 𝛽. Methods Enzymol. 1 : 149-1589
Christian, Gary D. 2014. Analytical Chemistry. United States of America : John Wiley &
Sons Inc.
Sheri, Madhu. 2016. Infinite Polyiodide Chains in The Pyrroloperylene iodine complex :
insights into the starch-iodine and perylene-iodine complexes. 55-8032-8035. DOI :
10.1002/anie.201601585
Wahyuni. 2015.Konversi Enzimatik Pengujia Aktivitas Enzim 𝛼-Amilase. Bandung : ITB
Yoo, Y.J. 1987. Comparison of 𝛼-Amilase activities from different assay methods. “141-
151”

XI. Answering the Question

1. Make the curve that figure the relation between pH and the velocity of enzimatic
reaction (∆A/min) !

Relation between pH and ΔA

0.18
0.16 y = 0.0128x + 0.0424
R² = 0.5245
0.14
0.12 0.135 0.133 0.136
0.121
ΔA/min

0.1
0.08 Series 1
0.06 Linear (Series 1)
0.04
0.02
0
0.007
0 2 4 6 8 10
pH

18 |
 2. Make the curve that figure the relation between concentration and the velocity of
enzimatic reaction (∆A/min) !

Relation between Concentration and ΔA

0.0160.015

0.014

0.012

0.01
0.007
ΔA/min

0.008
Y-Values
0.0060.005
Linear (Y-Values)
0.004
0.002
0.002
0
y = -7E-08x + 0.0075
0
R² = 0.3178
-20000 0 20000 40000 60000 80000 100000 120000
-0.002
Concentration (the number of dilution)

19 |
XII. Attachment
No Picture Description
1 Preparation of tools
and material

2 1 ml of starch + pH
solution (1, 3, 5, 7,
and 9) and 0,5 ml
salliva

3 1 ml of starch + pH
solution (1, 3, 5, 7,
and 9) and 0,5 ml
salliva is heated

4 Iodine and the solution

after iodine addition

5 6 ml aquades and it is
added to the solution

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 6 1 ml starch and
optimum pH (pH 9)
and 0,5 ml salliva
(10x. 100x, 103x,
104x, and 105x)

7 1 ml starch and
optimum pH (pH 9)
and 0,5 ml salliva
(10x. 100x, 103x,
104x, and 105x) is
heated in 37oC for 5
minutes and in 70oC
for 15 minutes
8 Measuring the
temperature

9 After iodine addition

21 |
22 |