A.

EXPERIMENT TITTLE :
Effect Of Enzyme Ph and Concentration on Enzyme Activity
B. EXPERIMENT START
Friday, September 3th, 2019 at 01.00 p.m
C. EXPERIMENT FINISH
Friday, September 3th, 2019 at 03.30 p.m
D. EXPERIMENT PURPOSE :
1. Prove that pH and concentration of the enzyme affect the enzyme activity
E. BASIC THEORY
1. Enzyme
a. Definition
Enzyme is a substance that acts as a catalyst in living organisms,
regulating the rate at which chemical reactions proceed without itself
being altered in the process (Eaton and Rogers, 2018). The biological
processes that occur within all living organisms are chemical reactions,
and most are regulated by enzymes. Without enzymes, many of these
reactions would not take place at a perceptible rate. Enzymes catalyze
all aspects of cell metabolism (Piergiovanni and Herrero-Martínez,
2019). This includes the digestion of food, in which
large nutrient molecules (such as proteins, carbohydrates, and fats) are
broken down into smaller molecules; the bconservation and
transformation of chemical energy; and the construction of cellular
macromolecules from smaller precursors. Many inherited human
diseases, such as albinism and phenylketonuria, result from a deficiency
of a particular enzyme (Emery, 1968).
Enzyme has some properties, include of physical and chemical
properties. Enzyme work properly in some of condition such as in
aqueous solution, normal pH, normal temperature, and certain
concentration of substrat. Enzyme work in specific thing. Enzyme has
molecular weight among 12.000 until more than 1 million. So, the size
of enzyme is bigger than the susbtrat (Lehninger, 1982).

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 Enzymes also have valuable industrial and medical applications. The
fermenting of wine, leavening of bread, curdling of cheese, and brewing
of beer have been practiced from earliest times, but not until the 19th
century were these reactions understood to be the result of the catalytic
activity of enzymes (Fischer, 2016). Since then, enzymes have assumed
an increasing importance in industrial processes that involve organic
chemical reactions. The uses of enzymes in medicine include killing
disease-causing microorganisms, promoting wound healing, and
diagnosing certain diseases.
b. Structure
Enzymes are proteins, and their function is determined by their complex
structure. The reaction takes place in a small part of the enzyme called
the active site, while the rest of the protein acts as
"scaffolding" (Boisseau and Lahmani, 2009). Enzymes are actually
made up of 1000s of amino acids that are linked in a specific way to form
different enzymes. The enzyme chains fold over to form unique shapes and
it is these shapes that provide the enzyme with its characteristic chemical
potential. Most enzymes also contain a non-protein component known as
the co-factor (Papachristodoulou, et al, 2014).
Many enzymes need cofactors (or coenzymes) to work properly. These
can be metal ions (such as Fe2+, Mg2+, Cu2+) or organic molecules
(such as haem, biotin, FAD, NAD or coenzyme A) (Lehninger, 1982).
Many of these are derived from dietary vitamins, which is why they are
so important. The complete active enzyme with its cofactor is called a
holoenzyme, while just the protein part without its cofactor is called the
apoenzyme (Kokate and Jajalpure, 2011).
c. Classification
The biochemical reactions occurring in the body are basically of 6 types
and the enzymes that bring about these reactions are named accordingly:
1) Oxidoreductase
These enzymes bring about oxidation and reduction reactions and
hence are called oxidoreductases (Maclean, 2016). In these reactions,

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 electrons in the form of hydride ions or hydrogen atoms are
transferred. When a substrate is being oxidized, these enzymes act as
the hydrogen donor. These enzymes are called dehydrogenases or
reductases. When the oxygen atom is the acceptor, these enzymes are
called oxidases.
2) Transferase
These enzymes are responsible for transferring functional groups
from one molecule to another (Maclean, 2016). Example: alanine
aminotransferase which shuffles the alpha‐amino group between
alanine and aspartate etc. Some transferases also transfer phosphate
groups between ATP and other compounds, sugar residues to form
disaccharides such as hexokinase in glycolysis.
3) Hydrolases
These enzymes catalyze reactions that involve the process of
hydrolysis. They break single bonds by adding water. Some
hydrolases function as digestive enzymes because they break the
peptide bonds in proteins (Maclean, 2016). Hydrolases can also be a
type of transferases as they transfer the water molecule from one
compound to another. Example: Glucose-6-phosphatase that removes
the phosphate group from glucose-6-phosphate, leaving glucose and
H3PO4.
4) Lyases
These enzymes catalyze reactions where functional groups are added
to break double bonds in molecules or where double bonds are
formed by the removal of functional groups (Campbell and Farrell,
2009). Example: Pyruvate decarboxylase is a lyase that removes
CO2 from pyruvate. Other examples include deaminases and
dehydratases.
5) Isomerases
These enzymes catalyze the reactions where a functional group is
moved to another position within the same molecule such that the
resulting molecule is actually an isomer of the earlier molecule

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 (Campbell and Farrell, 2009). Example: triosephosphate isomerase
and phosphoglucose isomerase for converting glucose 6-phosphate to
fructose 6-phosphate.
6) Ligases
These enzymes perform a function that is opposite to that of the
hydrolases. Where hydrolases break bonds by adding water, ligases
form bonds by removal of the water component. There are different
subclasses of ligases which involve the synthesis of ATP (Maclean,
2016)
d. Enzyme work and Enzyme Activity
Some enzymes help break large molecules into smaller pieces that are
more easily absorbed by the body (Patton and Thibodeau, 2017). Other
enzymes help bind two molecules together to produce a new molecule.
Enzymes are highly selective catalysts, meaning that each enzyme only
speeds up a specific reaction (Albert et al, 2013). The molecules that an
enzyme works with are called substrates. The substrates bind to a region
on the enzyme called the active site. There are two theories explaining
the enzyme-substrate interaction. In the lock-and-key model, the active
site of an enzyme is precisely shaped to hold specific substrates. In the
induced-fit model, the active site and substrate don't fit perfectly
together; instead, they both alter their shape to connect (Albert et al,
2013).
Theory 1: Lock and Key Hypothesis
This is the most accepted of the theories of enzyme action.

This theory states that the substrate fits exactly into the active site of the
enzyme to form an enzyme-substrate complex. This model also describes

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 why enzymes are so specific in their action because they are specific to
the substrate molecules.

Theory 2: Induced Fit Hypothesis

This is similar to the lock and key hypothesis. It says that the shape of the
enzyme molecule changes as it gets closer to the substrate molecule in
such a way that the substrate molecule fits exactly into the active site of
the enzyme.
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 (Cheswort et al, 2012).
Enzyme activity is a measure of the catalytic ability and there are two
methods to measure enzyme activity: one of them is to measure the
decrease in substrate concentration in a period of time, and the other is
to measure the increase in concentration of a product after a period of
time. The rate of a biochemical reaction at a g iven temperature and pH
depends on the enzyme concentration and the substrate concentration.
Provided the substrate concentration remains in excess, the initial rate is
directly proportional to the enzyme concentration (Soni, 2010).
e. Factors that Affect Enzyme Activity
Because active sites are finely tuned to help a chemical reaction happen,
they can be very sensitive to changes in the enzyme’s environment.
Factors that may affect the active site and enzyme function include:
1) Temperature

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 As the temperature rises, reacting molecules have more and more
kinetic energy. This increases the chances of a successful collision
and so the rate increases (Rosenmund, 1976). There is a certain
temperature at which an enzyme's catalytic activity is at its greatest
(see graph). This optimal temperature is usually around human
body temperature (37.5 oC) for the enzymes in human cells. Above
this temperature the enzyme structure begins to break down
(denature) since at higher temperatures intra- and intermolecular
bonds are broken as the enzyme molecules gain even more kinetic
energy.

2) The effect of pH
pH can also affect enzyme function. Active site amino acid
residues often have acidic or basic properties that are important for
catalysis. Changes in pH can affect these residues and make it hard
for substrates to bind. Enzymes work best within a certain pH
range, and, as with temperature, extreme pH values (acidic or
basic) can make enzymes denature.

3) The effectf of enzyme concentration

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 Increasing enzyme concentration will speed up the reaction, as long
as there is substrate available to bind to. Once all of the substrate is
bound, the reaction will no longer speed up, since there will be
nothing for additional enzymes to bind to.

4) The effect of substrate concentration

Increasing substrate concentration also increases the rate of
reaction to a certain point. Once all of the enzymes have bound,
any substrate increase will have no effect on the rate of reaction, as
the available enzymes will be saturated and working at their
maximum rate.

5) Inhibitors
Presence of certain substances that inhibit the action of a particular
enzyme. This occurs when the inhibiting substance attaches itself to
the active site of the enzyme thereby preventing the substrate
attachment and slows down the process. Inhibitors that occupy the
active site and prevent a substrate molecule from binding to the
enzyme are said to be active site-directed (or competitive, as they
'compete' with the substrate for the active site). Inhibitors that
attach to other parts of the enzyme molecule, perhaps distorting its
shape, are said to be non-active site-directed (or non competitive).

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2. Amylase Enzyme
Amylase is any member of a class of enzymes that catalyze
the hydrolysis (splitting of a compound by addition of a water molecule)
of starch into smaller carbohydrate molecules such
as maltose (a molecule composed of two glucose molecules) (Yamamoto,
1995). Two categories of amylases, denoted alpha and beta, differ in the
way they attack the bonds of the starch molecules. 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, whereas pancreatic amylase is secreted by
the pancreas into the small intestine. Ptyalin is mixed with food in the
mouth, where it acts upon starches. Although the food remains in the mouth
for only a short time, the action of ptyalin continues for up to several hours
in the stomach—until the food is mixed with the stomach secretions, the
high acidity of which inactivates ptyalin (Pandey et al, 2006). Ptyalin’s
digestive action depends upon how much acid is in the stomach, how
rapidly the stomach contents empty, and how thoroughly the food has
mixed with the acid (Pandey et al, 2006). Under optimal conditions as
much as 30 to 40 percent of ingested starches can be broken down to
maltose by ptyalin during digestion in the stomach (Butterly and Shepherd,
2010). Because it can act anywhere on the substrate, α-amylase tends to be
faster-acting than β-amylase. In animals, it is a major digestive enzyme,
and its optimum pH is 6.7–7.0. In human physiology, both the salivary and
pancreatic amylases are α-amylases (Yamamoto, 1995).
The activity of amylase can be observed by using iodine. Because iodine
reacts with starch to form a dark brown/purple color (Harisha, 2005). As
amylase breaks down starch, less and less starch will be present and the
color of the solution (if iodine is added) will become lighter and lighter.
The extent of the hydrolysis depends on how long it is allowed to react –if
the starch is hydrolyzed completely, the resulting product is glucose
(Benkeblia, 2014). You will test for the presence or absence of starch in the

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 solutions using iodine (I2). Iodine forms a blue to black complex with
starch, but does not react with glucose.
3. Starch (Amylum)
Starch is a white, granular, organic chemical that is produced by all green
plants. Starch is a soft, white, tasteless powder that is insoluble in
cold water, alcohol, or other solvents. The basic chemical formula of the
starch molecule is (C6H10O5)n. Starch is
a polysaccharide comprising glucose monomers joined in α 1,4 linkages.
The simplest form of starch is the linear polymer amylose; amylopectin is
the branched form (Chandrasekaran, 2015).
Starch is manufactured in the green leaves of plants from excess glucose
produced during photosynthesis and serves the plant as a reserve food
supply (Macmillan, 1999). Starch is stored in chloroplasts in the form of
granules and in such organs as the roots of the tapioca plant; the tuber of
the potato; the stem pith of sago; and the seeds of corn, wheat, and rice.
When required, starch is broken down, in the presence of certain enzymes
and water, into its constituent monomer glucose units, which diffuse from
the cell to nourish the plant tissues (Hodge, 2017). In humans and other
animals, starch is broken down into its constituent sugar molecules, which
then supply energy to the tissues (Chandrasekaran, 2015). Most
commercial starch is made from corn, although wheat, tapioca,
and potato starch are also used. Commercial starch is obtained by crushing
or grinding starch-containing tubers or seeds and then mixing the pulp with
water; the resulting paste is freed of its remaining impurities and then dried.
Aside from their basic nutritional uses, starches are used in brewing and as
thickening agents in baked goods and confections. Starch is used
in paper manufacturing to increase the strength of paper and is also used in
the surface sizing of paper. Starch is used in the manufacture of corrugated
paperboard, paper bags and boxes, and gummed paper and tape (Hodge,
2017). Large quantities of starch are also used in the textile industry as
warp sizing, which imparts strength to the thread during weaving.
4. Spectrophotometry UV-Vis

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 Spectrophotometry is a method to measure how much a chemical
substance absorbs light by measuring the intensity of light as a beam of
light passes through sample solution (Akio, 1989). The basic principle is
that each compound absorbs or transmits light over a certain range of
wavelength. This measurement can also be used to measure the amount of a
known chemical substance results. Spectrophotometry is one of the most
useful methods of quantitative analysis in various fields such as chemistry,
physics, biochemistry, material and chemical engineering and clinical
applications (Akio, 1989). A spectrophotometer is an instrument that
measures the amount of photons (the intensity of light) absorbed after it
passes through sample solution. With the spectrophotometer, the amount of
a known chemical substance (concentrations) can also be determined by
measuring the intensity of light detected. Depending on the range of
wavelength of light source. In visible spectrophotometry, the absorption or
the transmission of a certain substance can be determined by the observed
color. For instance, a solution sample that absorbs light over all visible
ranges (i.e., transmits none of visible wavelengths) appears black in theory.
On the other hand, if all visible wavelengths are transmitted (i.e., absorbs
nothing), the solution sample appears white. If a solution sample absorbs
red light (~700 nm), it appears green because green is the complementary
color of red. Visible spectrophotometers, in practice, use a prism to narrow
down a certain range of wavelength (to filter out other wavelengths) so that
the particular beam of light is passed through a solution sample.

F. TOOLS AND MATERIALS

1. Tools
a. Test tube 6 pieces
b. Volumetric Flask 10mL 1 piece
c. Water Bath 1 piece
d. Beaker Glass 1 piece
e. Rack 1 piece
f. Pipette 5 pieces
g. Graduated cylinder 10 mL 1 piece

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 h. Tripod 1 piece
i. Gauze 1 piece
j. Thermometer 1 piece
k. Spectrophotometry UV-Vis 1 piece
l. Cuvette 1 piece
2. Materials
a. Saliva 5 mL
b. Aquades 250 mL
c. Iodine 24 drops
d. Starch 12 mL
G. LANES WORK
1. Dilution Enzyme Solution
a. Dilution 10 time

1 mL Saliva

1.Dilute 10 times

Enzyme With 10 Times

Dilution

b. (Repeat for 20, 30, 40, 50 times)

2. Effect of pH on Enzyme Activity
a. Blanko
1 mL of starch solution

1. Put in a test tube

2. Added 0.5 mL aquades
3. Left for 3 minutes at 37˚ C
4. Added 2 drops of Iodine solution
5. Added 6 mL aquades
6. Read absorbance at wavelength 680 nm

Absorbant

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 b. Test

1 mL of starch + 1 mL solution pH (1, 3, 5,

7, 9)
1. Put in a test tube
2. Added 0.5 mL enzyme 10 x dilution
3. Left for 3 minutes at 37˚ C
4. Added 2 drops of Iodine solution
5. Added 6 mL aquades
6. Read absorbance at wavelength 680 nm

Absorbant

3. Effect of Concentration on Enzyme Activity

a. Blanko

1 mL of starch solution + 1 mL of pH
optimum
1. Put in a test tube
2. Added 0.5 mL aquades
3. Left for 3 minutes at 37˚ C, and 3 minutes >60˚C
4. Added 2 drops of Iodine solution
5. Added 6 mL aquades
6. Read absorbance at wavelength 680 nm

Absorbant

b. Test

1 mL of starch solution + 1 mL of pH
optimum
1. Put in a test tube
2. Added 0.5 mL enzyme 10-50 x dilution
3. Left for 3 minutes at 37˚ C, and 3 minutes >60˚C
4. Added 2 drops of Iodine solution
5. Added 6 mL aquades
6. Read absorbance at wavelength 680 nm

Absorbant

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H. EXPERIMENT RESULT

Experiment Result
No. Experiment Procedure Assumption/Reaction Conclusion
Before After
1. Dilution of Saliva  Saliva: colorless 1 mL Saliva +
solution aquadest
1 mL Saliva
 Dilution 10 times :
colorless solution
1.Dilute 10 times
 Dilution 20 times :
colorless solution
Enzyme With 10 Times
 Dilution 30 times :
Dilution
colorless solution

Repeat the step by changing the  Dilution 40 times :

dilution into 20, 30, 40 and 50. colorless solution
 Dilution 50 times :
colorless solution

2. Effect of pH on Enzyme Activity  Starch solution:  Starch solution +  pH affect

white turbid aquades: 2 layers, enzyme
a. Blanco solution turbid solution activity
 pH
 Aquades: colorless  Heat 37oC : still 2

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 Experiment Result
No. Experiment Procedure Assumption/Reaction Conclusion
Before After

1 mL of starch solution solution layers optimum in

this
 + 2 drops of I2: experiment
1.Put in a test tube
dark blue solution is 9
2.Added 0.5 mL aquades
3.Left for 3 minutes at 37˚C  + 6 mL aquades:
4.Added 2 drops of Iodine blue solution
solution (++++)
5.Added 6 mL aquades  Absorbance: 1.875
6.Read absorbance at
wavelength 680 nm

Absorbance

b. Test  Starch : white  Starch + pH

turbid solution solution for each
test tube : 2 layers
 pH solution: solution (turbid
colorless solution lower, colorless
 Enzyme: colorless upper)
solution  + enzyme: 2 layers

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 Experiment Result
No. Experiment Procedure Assumption/Reaction Conclusion
Before After

1 mL of starch + 1 mL solution  Aquades: colorless  Heat for 3 minutes

solution at 37oC: still two
pH (1, 3, 5, 7, 9)
layers solution
 Starch: turbid
1.Put in a test tube solution  + 2 drops of
2.Added 0.5 mL enzyme iodine for each
10x dilution  Iodine: brownish test tube : dark
3.Left for 3 minutes at yellow solution blue solution
37˚C
4.Added 2 drops of Iodine  + 6 mL aquadest
solution pH 1: blue
5.Added 6 mL aquades solution
6.Read absorbance at (++)
wavelength 680 nm
pH 3: blue
Absorbant solution
(++++)
pH 5: blue
solution
(+++)

(Soewarto, 2000)
pH optimum amylase

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 Experiment Result
No. Experiment Procedure Assumption/Reaction Conclusion
Before After
pH 7: blue based on theory : 6,8-7.0
solution
(++)
pH 9: blue
solution (+)
 Absorbance (A)
pH 1: 0.619
pH 3: 1.512
(Soewarto, 2000)
pH 5: 1.106
pH 7: 1.055
pH 9: 0.227 (pH
optimum)
 ∆A
pH 1: 1.256
pH 3: 0.363
pH 5: 0.769
pH 7: 0.82

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 Experiment Result
No. Experiment Procedure Assumption/Reaction Conclusion
Before After
pH 9: 1.648
3. Effect of Concentration on Enzyme  Starch: white  Starch + pH 9 Based on thix
Activity turbid solution solution: 2 layers experiment,
(turbid lower, amylase
a. Blanco  pH 9 solution: colorless upper) enzyme
colorless solution concentration
1 mL of starch solution + 1 mL  + aquades: still 2
 Aquades: colorless affect the
of pH optimum layers enzyme
solution
 Heated for 3 activity, higher
1.Put in a test tube  Iodine: brownish minutes at 37oC : concentration
2.Added 0.5 mL aquades yellow solution still 2 layers also higher its
3.Left for 3 minutes at activity.
37˚C, and 3 minutes  Heated for 3
>60˚C minutes at >60oC :
4.Added 2 drops of Iodine still 2 layers
solution  + 2 drops of
5.Added 6 mL aquades iodine: blue
6.Read absorbance at solution (++++)
wavelength 680 nm
 + aquades: pale
Absorbant blue solution (++)
 Absorbance: 0.460
(Soewarto, 2000)

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 Experiment Result
No. Experiment Procedure Assumption/Reaction Conclusion
Before After

b. Test  Starch solution:  Starch + pH

white turbid optimum: 2 layers
solution (turbid lower,
colorless upper) (Soewarto, 2000)
 pH 9 solution:
colorless solution  + 0.5 mL enzyme:
still 2 layers
 Enzyme (10x-50x
dilution): colorless  Heated for 3
solution minutes at 37oC :
still 2 layers
 Aquades: colorless
solution  Heated for 3
minutes at >60oC :
 Iodine: brownish
yellow solution  + iodine : all test
tube blue solution

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 Experiment Result
No. Experiment Procedure Assumption/Reaction Conclusion
Before After

1 mL of starch solution + 1 mL  + 6 mL aquades

of pH optimum 10x: turbid blue
solution (++)
1.Put in a test tube
2.Added 0.5 mL enzyme 20x: turbid blue
10-50x dilution solution (+)
3.Left for 3 minutes at 30x: turbid blue
37˚C, and 3 minutes solution (+)
>60˚C
40x: turbid blue
4.Added 2 drops of Iodine
solution
solution
5.Added 6 mL aquades 50x: turbid blue
6. Read absorbance at solution
wavelength 680 nm  Absorbance (A)
Absorbant 10x: 0.077
20x: 0.097
30x: 0.298
40x: 0.405
50x: 0.377
 ∆A

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 Experiment Result
No. Experiment Procedure Assumption/Reaction Conclusion
Before After
10x: 0.383
20x: 0.363
30x: 0.162
40x: 0.055
50x: 0.083

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I. ANALYSIS AND EXPLANATION
This experiment aims to prove that pH and concentration of the
enzyme affect the enzyme activity. Enzyme is a substance that acts as
a catalyst in living organisms, regulating the rate at which chemical
reactions proceed without itself being altered in the process (Eaton and Rogers,
2018). The biological processes that occur within all living organisms
are chemical reactions, and most are regulated by enzymes. Without enzymes,
many of these reactions would not take place at a perceptible rate. Enzymes
catalyze all aspects of cell metabolism (Piergiovanni and Herrero-Martínez,
2019).
Enzyme activity is the number of moles of substrateconverted per unit
time. Under optimum conditions, the enzymatic reaction rate will work
optimally, so that more products are obtained. Concentration and pH affect the
enzyme activity, because every enzyme has optimal pH to optimization the
enzyme activity. In this experiment we use human saliva as a source of
amylase enzyme. Amylase is a member of a class of enzymes that catalyze
the hydrolysis (splitting of a compound by addition of a water molecule)
of starch into smaller carbohydrate molecules such
as maltose (a molecule composed of two glucose molecules) (Yamamoto,
1995).
The principle of this experiment are hydrolysis of starch (amylum) with
amylase as catalyst and the reaction form complex amylum-iod, to detect the
rest of amylum in the sample which doesn’t hydrolyzed yet. Starch hydrolized
become glucose by amylase enzime. Enzyme is protein, so it can be
denaturated by temperature and pH (Lehninger, 1982). In this experiment, we
use two kind of independent variabel to know and determine those influence in
enzyme activities, which are pH and concentration. Based on theory, the
optimum pH of amylase enzime is 6,8-7,0. Other, the increasing of enzyme
concentration will also increase the enzyme activities. Increasing the enzyme
activities shown with increasing A (Soewoto, 2000).
To know the effect of pH and enzyme concentration on enzyme activity
we use spectrophotometry uv-vis as an instrument. Spectrophotometry is a

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method to measure how much a chemical substance absorbs light by measuring
the intensity of light as a beam of light passes through sample solution (Akio,
1989). In spektrophotometry Uv-Vis, there are two kinds of light wave which
are UV and visible light. Ultraviolet (UV) has wavelenght for about 200-400
nm while Visible light has wavelength for about 400-750 nm. Complex
amylum-iod include in visible light because the compound forward the
complementary blue color to the eye. The maximum wavelength of complex
amylum-iod is 680 nm (Soewoto, 2000).
This experiment has 3 step, the first is dilution enzyme solution, the
second step is effect of pH in enzyme activity and the third step is effect of
concentration in enzyme activity.
1. Dilution enzyme solution
The aims of this experiment is to decrease the enzyme
concentration. In step number 3, we want to prove that the concentration of
enzyme affect the enzyme activity, so that we need various of enzyme
concentration through dilution the enzyme. Enzyme dilution is also done
to reduce the enzyme concentration so that the analyzed enzyme can meet
the Limit of Detection (LOD) on UV-Vis specrophotometry, which is
0.09-1 μg/dL (Beers, 1951). In this experiment, we will use 5 different of
enzyme concentration using dillution of 10x, 20x, 30x, 40x, and 50x.
First, we got the amylase enzyme by human saliva, because based
on theory, amylase enzyme contain in mouth and pancreas. So we use
human saliva as the source of amylase enzyme. We need more about 5 mL
of human saliva. Then we pour 1 mL of saliva into 10 mL of volumetric
flask, then we adding the aquades until it reach the meniscus line. Then we
shake it to make the solution homogeneous. After that, we pour the
solution to the beaker glass, then noted it as enzyme solution with 10x
dilution. After that, repeat the step of pouring 1 mL of saliva in into 10 mL
of volumetric flask then we adding the aquades until it reach the meniscus
line, shake it until homogeneous. After that, pour the enzyme into beaker
glass then we adding again the aquades into the volumetric flask that has
been used until it reach a miniscus and shake it again, the solution noted as

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 20x dilution. So, for the 30x dilution we add we added aquadest twice,
thrice times, and four times. The color of the solution of all diluted enzyme
is colorless with bubble.
2. Effect of pH in enzyme activity
This experiment aims to prove that pH affect the enzyme activity.
Extreme high or low pH (too acid or too base) will affect of losing enzyme
function because of denaturation of enzyme because basically enzyme is
protein (Lehninger, 1982). Each enzyme has their own optimum pH. For
amylase enzyme, the optimum pH is 6,8-7,0 (Soewoto, 2000). pH is also
a factor in the stability of enzymes. As with activity, for each
enzyme there is also a region of pH optimal stability.
a. Blank solution
A blank solution is solution which has all the component same with
sample solution except the analyte. Blank solution is used as
comparation. Blank solution which contain all the component same
with sample but except the analyte will shows the difference in changes
that occur in a solution that does not contain analytes with the solution
that contain analytes. In this experiment, the blank solution doesn’t
contain enzyme, so that we will know the enzyme activities by
comparing the condition before and after addition of enzyme in cetain
pH. The difference result of blank solution absorbance and sample
solution absorbance will show the effect of pH in enzyme activities.
Blank solution also used to calculate the value of A where it can
show how much the amount of amylum which already degrade become
glucose and the remaining amylum.
The first step of this experiment is pouring 1 mL of the starch solution
into the test tube, starch solution is turbid, then added with 0.5 mL
aquades. The function of adding 0.5 mL of aquadest is to replace the
enzyme solution in sample solution. So, the total volume of blanco and
enzyme solution test will same. After added 0.5 mL aquadest, solution
is still turbid. Then left for 3 minutes at 37oC, this step aims to make
the same treatment between blank solution and sample solution so the

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result will accurate and precise. After that, we added 2 drops of iodine,
and the solution become dark blue. The function of I2 is to indicate the
amount of starch in the blanco solution. So that, the rest of starch that
didn’t hydrolized by amylase enzyme can be detected. In blank solution
which doesn’t contain amylase enzyme, the color of the solution is dark
because the amylum doesn’t hydrolyzed properly without the existence
of enzyme. Iodine use as the indicator to showing the presence of
amylum and to give color for analysis using spectrophotometry uv-vis.
The solution which become dark blue (++++) shows that complex
amylum-iod are formed. The reaction is :

(Soewarto, 2000)

Then we added 6 mL of aquadest, and the color of the solution become

faded. This step aims to to make the blanco solution meet the LOD of
the spectrophotometry uv-vis, spectrophotometry uv-vis has LOD in
range of 0,1-1. If the sample is too concentrated, the detector can’t
differentiate incoming rays and transmitted rays, so the detector will
read the absorbance using prediction mode. If the result of absorbance
are not in range of 0,1-1 means that the result is inaccurate and not valid
data (Underwood, 1998). The blank solution then measured for
absorbance at a wavelength of 680 nm using spectrophotometry uv-vis.
Spectrophotometry is a method to measure how much a chemical
substance absorbs light by measuring the intensity of light as a beam of

24 | P a g e
 light passes through sample solution (Akio, 1989). The basic principle
is that each compound absorbs or transmits light over a certain range of
wavelength. This measurement can also be used to measure the amount
of a known chemical substance results. Each substance has a specific
properties to wavelength, the wavelength of 680 nm is the maximum
wavelength of the iod-starch complex so that all iod-starch complexes
will be absorbed by the detector. The absorbance value of the blanco
solution is 1.875. This result is higher than LOD it means that there’s
deviation in the absorbance result. This error can be caused by chemical
factors, namely deposition may occur while waiting for the wavelength
test using spectronic-20 so that the absorbance that is read is absorbing
the sediment. It is possible that the cuvette is contaminated with
impurities so that the absorbance of the impurities is read. But, for this
experiment to knowing the effect of pH on enzyme activity it is stil
permitted because this data still can be used to comparing and knowing
the enzyme activity.

b. Test
In this step we will use five different pH namely 1, 3, 5, 7, and 9 which
already provided in laboratory of biochemistry. This experiment also
used to determine the pH optimum of the enzyme sample. The first step
of this experiment is preparing five different test tube, and for each test
tube poured with 1 mL of starch and 1 mL solution pH (1, 3, 5, 7 and
9). The color of all the pH solution is colorless, so after the pH added
in 1 mL of starch it becomes colorless too. Then for each test tube,
added with 0.5 mL amylase enzyme (saliva) 10 x dilution, this addition
used as control variable of this experiment. Amylase is a member of a
class of enzymes that catalyze the hydrolysis (splitting of
a compound by addition of a water molecule) of starch into smaller
carbohydrate molecules such as maltose (a molecule composed of
two glucose molecules) (Yamamoto, 1995). So in this experiment
amylase will catalyzed the hydrolysis of amylum to become smaller
molecule (glucose). After that left for 3 minutes at 37˚ C using

25 | P a g e
waterbath, this step is done because 37˚C is the optimum temperature of
amylase enzyme to work properly. If the temperature lower than 37˚C
the enzyme won’t work properly, but if the temperature higher than
37˚C the enzyme will denaturated because basically enzyme formed
from protein which can be denaturated in high temperature. The
warming process need 3 minutes because hydrolisis needs several time
to break the linkage. So, waiting 3 minutes is to make sure that the
hydrolysis of amylum with amlyase enzyme as catalyst can work
properly. The hydrolysis reaction is :

Then added 2 drops of Iodine solution for each test tube, then the color
of the solution change become dark blue. The function of I2 is to
indicate the amount of starch in the blanco solution. So that, the rest of
starch that didn’t hydrolized by amylase enzyme can be detected. Two
categories of amylases, denoted alpha and beta, differ in the way they
attack the bonds of the starch molecules. 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. As amylase breaks down starch, less and less starch
will be present and the color of the solution will become lighter and
lighter using iodine indicator. The extent of the hydrolysis depends on
how long it is allowed to react –if the starch is hydrolyzed completely,
the resulting product is glucose (Benkeblia, 2014). We test for the
presence or absence of starch in the solutions using iodine (I2). Iodine
forms a blue to black complex with starch, but does not react with
glucose. The reaction is :

26 | P a g e
(Soewarto, 2000)
After that added 6 mL aquades for each test tube, and the color of the
solution become faded one by one. This step aims to to make the
sample solution meet the LOD of the spectrophotometry uv-vis,
spectrophotometry uv-vis has LOD in range of 0,1-1. If the sample is
too concentrated, the detector can’t differentiate incoming rays and
transmitted rays, so the detector will read the absorbance using
prediction mode. If the result of absorbance are not in range of 0,1-1
means that the result is inaccurate and not valid data (Underwood,
1998). The sample solution then measured for absorbance at a
wavelength of 680 nm using spectrophotometry uv-vis.
Spectrophotometry is a method to measure how much a chemical
substance absorbs light by measuring the intensity of light as a beam of
light passes through sample solution (Akio, 1989). The basic principle
is that each compound absorbs or transmits light over a certain range of
wavelength. This measurement can also be used to measure the amount
of a known chemical substance results. Each substance has a specific
properties to wavelength, the wavelength of 680 nm is the maximum
wavelength of the iod-starch complex so that all iod-starch complexes
will be absorbed by the detector. The absorbance value of sample
solution and the color change of five sample solution with different pH
solution is listed in this following table :

27 | P a g e
 Ph Color change Absorbance (absorbance blank –
Solution (A) absorbance sample)

Blank Blue solution 1.875

-
(+++++)
1 Blue solution 0.619
1.256
(++)
3 blue solution 1.512
0.363
(++++)
5 blue solution 1.106
0.769
(+++)
7 blue solution 1.055
0.820
(++)
9 blue solution (+) 0.227 1.648

Based on the data, we know that the blank solution which doesn’t added
with pH solution and enzyme has higher absorbance value, it is because
the amylum in the blank solution doesn’t hydrolyzed yet so that it has
higher starch content. Then, from the low pH until high pH, the color of
blue become more fadded. This means that the amylum contain in the
sample are decrease. But in pH 1, we get the result that the color change
and absorbance is lower than pH 3, this result is not valid because
increasing pH should be followed by decreasing blue color in solution.
This is may caused by the contaminant substance in the sample which
can affect the result. Another possibility is may caused by damaged pH
solution or other reagent. If the rest of amylum in the sample is little, it
means that the enzyme in those test tube are optimum and active. The
decreasing of blue color also impact in decreasing the value of
absorbance. The value of absorbance show us the amount of remaining
amylum. The lowest remaining amylum is in pH 9 with minimum
absorbance value 0.227.
The graphic which based on theory is :

28 | P a g e
 optimum

𝑝𝐻

The graphic based on experiment result :

The Influence of pH to Enzyme Activity

2

1.5
Absorbance

0.5 Absorbance

0
0 2 4 6 8 10
pH

From the data, we can get the value of enzyme activity based on the
graphic. According to the theory, the graphic of enzyme activity can be
know by creating graphic pH vs absorbance.

optimum

𝑝𝐻
The graphic based on the experiment result is:

29 | P a g e
 The Influence of pH to Enzyme Activity
2

1.5

1
delta absorbance
0.5

0
0 5 10
pH

Based on all the result, we can conclude that the optimum pH of this
experiment is 9.0. Because at pH 9 it has the smallest absorbance value,
which means that at pH 9 absorption by the spectrophotometer is
getting smaller because many starches have been hydrolyzed into
glucose. And from the graphic pH vs absorbance we know how much
amylum which has been hydrolyzed, enzyme activity higher when the
pH is 9.0 which means that at pH 9 the amylum which hydrolyzed to
become glucose has higher value. This result is not accordance with the
theory which state that the optimum pH of amylase enzyme is 6.8-7.0.
This different result can caused of many factor, the main factors is
unhealthy condition of the volunteers when give the saliva sample.
Body conditions also affect the work of enzymes. In unhealthy body
conditions, enzymes cannot work as they should (Pratama, et al., 2012).
Another possibilities are the existence of contaminan substance which
can cause the accumulation of absorbance results (Underwood and Day,
2002).
3. Effect of concentration in enzyme activity
This experiment aim to prove that the concentration of enzyme affect
enzyme activity. Based on the theory the effect of enzyme concentration on
enzyme work is that the speed of the reaction will increase if the
concentration of the enzyme is added to a chemical reaction (Poedjiadi,
1994). Increasing the concentration of enzymes will increase the speed of
enzymatic reactions. It can be conclude that the speed of the enzymatic
reaction (v) is directly proportional to the concentration of the enzyme [E].

30 | P a g e
The greater the concentration of the enzyme, the faster the reaction. The
graphic that show eznyme activity vs concentration are :

𝐴 𝐴

Dilution Concentratio
n

a. Blank solution
A blank solution is solution which has all the component same with
sample solution except the analyte. Blank solution is used as
comparation. Blank solution which contain all the component same
with sample but except the analyte will shows the difference in changes
that occur in a solution that does not contain analytes with the solution
that contain analytes. In this experiment, the blank solution doesn’t
contain enzyme, so that we will know the enzyme activities by
comparing the condition before and after addition of enzyme in cetain
concentration. The difference result of blank solution absorbance and
sample solution absorbance will show the effect of concentration in
enzyme activities. Blank solution also used to calculate the value of
A where it can show how much the amount of amylum which already
degrade become glucose and the remaining amylum.
The first step of this experiment is pouring 1 mL of the starch solution
and 1 mL of pH optimum into the test tube, starch solution is turbid and
pH solution is colorless. The function of adding the pH optimum is as
the control variable, so that the enzyme can work properly to degrade
the amylum. Then added with 0.5 mL aquades. The function of adding
0.5 mL of aquadest is to replace the enzyme solution in sample
solution. So, the total volume of blanco and enzyme solution test will
same. After added 0.5 mL aquadest, solution is still turbid. Then left for
3 minutes at 37oC, this step aims to make the same treatment between

31 | P a g e
blank solution and sample solution so the result will accurate and
precise. After that, we heating the all the solution sample in temperature
which higher than 60oC. This purpose is to inactive the enzyme in
enzyme sample tested. Temperature higher than 60oC use to denaturate
proteins and to stop or inhibit the activity of our enzymes. Enzymes
including proteins and proteins can be denatured. Denaturation is a
change in protein structure from high to low levels. If the enzyme
continues to work, there will be residual starch when added to iod then
complex starch iod so that the color will not appear, can not be
absorbed, and we does not get the data, it will make the purpose of the
experiment is not achieved. After that we added 2 drops of iodine, and
the solution become dark blue. The function of I2 is to indicate the
amount of starch in the blanco solution. So that, the rest of starch that
didn’t hydrolyzed can be detected. In blank solution which doesn’t
contain amylase enzyme, the color of the solution is dark because the
amylum doesn’t degrade by the enzyme. Iodine use as the indicator to
showing the presence of amylum and to give color for analysis using
spectrophotometry uv-vis. The solution which become dark blue
(++++) shows that complex amylum-iod are formed. The reaction is :

(Soewarto, 2000)

Then we added 6 mL of aquadest, and the color of the solution become

faded. This step aims to to make the blanco solution meet the LOD of

32 | P a g e
 the spectrophotometry uv-vis, spectrophotometry uv-vis has LOD in
range of 0,1-1. If the sample is too concentrated, the detector can’t
differentiate incoming rays and transmitted rays, so the detector will
read the absorbance using prediction mode. If the result of absorbance
are not in range of 0,1-1 means that the result is inaccurate and not valid
data (Underwood, 1998). The blank solution then measured for
absorbance at a wavelength of 680 nm using spectrophotometry uv-vis.
Spectrophotometry is a method to measure how much a chemical
substance absorbs light by measuring the intensity of light as a beam of
light passes through sample solution (Akio, 1989). The basic principle
is that each compound absorbs or transmits light over a certain range of
wavelength. This measurement can also be used to measure the amount
of a known chemical substance results. Each substance has a specific
properties to wavelength, the wavelength of 680 nm is the maximum
wavelength of the iod-starch complex so that all iod-starch complexes
will be absorbed by the detector. The absorbance value of the blanco
solution is 0.460.

b. Test
In this step we will use five different concentration according to the
first step (dilution) namely 10x, 20x, 30x, 40x and 50x dilution. The
first step of this experiment is preparing five different test tube, and for
each test tube poured with 1 mL of starch solution and 1 mL of pH
optimum (pH 9.0). The color of all the solution is colorless, after the
pH added in 1 mL of starch it becomes colorless too. Then for each test
tube, added with 0.5 mL amylase enzyme (saliva) with different
concentration, this addition used as manipulation variable of this
experiment, so that we can know the effect of concentration enzyme in
enzyme activity. Amylase is a member of a class of enzymes that
catalyze the hydrolysis (splitting of a compound by addition of a water
molecule) of starch into smaller carbohydrate molecules such
as maltose (a molecule composed of two glucose molecules)
(Yamamoto, 1995). So in this experiment amylase will catalyze the

33 | P a g e
hydrolysis of amylum to become smaller molecule (glucose).
According to the theory the effect of enzyme concentration on enzyme
work is that the speed of the reaction will increase if the concentration
of the enzyme is added to a chemical reaction (Poedjiadi, 1994).
According to Hafiz Soewoto (2000) Increasing the concentration of
enzymes will increase the speed of enzymatic reactions. It can be said
that the speed of the enzyme reaction ik (v) is directly proportional to
the concentration of the enzyme [E]. The greater the enzyme
concentration, the faster the reaction, so when the concentration of the
enzyme is higher, the less residual amylum that has not been
hydrolyzed . After that left for 3 minutes at 37˚ C using waterbath, this
step is done because 37˚C is the optimum temperature of amylase
enzyme to work properly. If the temperature lower than 37˚C the
enzyme won’t work properly, but if the temperature higher than 37˚C
the enzyme will denaturated because basically enzyme formed from
protein which can be denaturated in high temperature. The warming
process need 3 minutes because hydrolisis needs several time to break
the linkage. So, waiting 3 minutes is to make sure that the hydrolysis
of amylum with amlyase enzyme as catalyst can work properly. The
hydrolysis reaction is :

After that, we heating the all the solution sample in temperature which
higher than 60oC. This purpose is to inactive the enzyme in enzyme
sample tested. Temperature higher than 60oC use to denaturate proteins
and to stop or inhibit the activity of our enzymes. Enzymes including
proteins and proteins can be denatured. Denaturation is a change in
protein structure from high to low levels. If the enzyme continues to
work, there will be residual starch when added to iod then complex
starch iod so that the color will not appear, can not be absorbed, and

34 | P a g e
we does not get the data, it will make the purpose of the experiment is
not achieved. Then added 2 drops of Iodine solution for each test tube,
then the color of the solution change become dark blue. The function
of I2 is to indicate the amount of starch in the blanco solution. So that,
the rest of starch that didn’t hydrolized by amylase enzyme can be
detected. Two categories of amylases, denoted alpha and beta, differ in
the way they attack the bonds of the starch molecules. 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. As amylase
breaks down starch, less and less starch will be present and the color of
the solution will become lighter and lighter using iodine indicator. The
extent of the hydrolysis depends on how long it is allowed to react –if
the starch is hydrolyzed completely, the resulting product is glucose
(Benkeblia, 2014). We test for the presence or absence of starch in the
solutions using iodine (I2). Iodine forms a blue to black complex with
starch, but does not react with glucose. The reaction is :

(Soewarto, 2000)
After that added 6 mL aquades for each test tube, and the color of the
solution become faded one by one. This step aims to to make the
sample solution meet the LOD of the spectrophotometry uv-vis,
spectrophotometry uv-vis has LOD in range of 0,1-1. If the sample is
too concentrated, the detector can’t differentiate incoming rays and

35 | P a g e
 transmitted rays, so the detector will read the absorbance using
prediction mode. If the result of absorbance are not in range of 0,1-1
means that the result is inaccurate and not valid data (Underwood,
1998). The sample solution then measured for absorbance at a
wavelength of 680 nm using spectrophotometry uv-vis.
Spectrophotometry is a method to measure how much a chemical
substance absorbs light by measuring the intensity of light as a beam of
light passes through sample solution (Akio, 1989). The basic principle
is that each compound absorbs or transmits light over a certain range
of wavelength. This measurement can also be used to measure the
amount of a known chemical substance results. Each substance has a
specific properties to wavelength, the wavelength of 680 nm is the
maximum wavelength of the iod-starch complex so that all iod-starch
complexes will be absorbed by the detector. The absorbance value of
sample solution and the color change of five sample solution with
different concentration enzyme is listed in this following table :
Dilution Color change Absorbance (A) (absorbance blank –
absorbance sample)

Blank Blue solution 0.460

-
(+++)
10 x turbid blue
0.383
solution (++) 0.077
20x turbid blue
0.363
solution (+) 0.097
30x turbid blue
0.171
solution (+) 0.289
40x turbid blue
0.055
solution 0.405
50x turbid blue
0.083
solution 0.377

Based on the data, we know that the blank solution which doesn’t
added with enzyme has higher absorbance value, it is because the

36 | P a g e
amylum in the blank solution doesn’t hydrolyzed yet so that it has
higher starch content. Then, from the higher concentration (10x
dilution) and the lower concentration (50x dilution) the color of blue
become more fadded and the lowest absorbance value is at 10x
dillution. It means that at 10x dilution, amylase has minimum value,
because it is already hydrolyzed to become glucose. If the rest of
amylum in the sample is little, it means that the enzyme in those test
tube are optimum and active. The value of absorbance show us the
amount of remaining amylum. The lowest remaining amylum is in 10x
dilution sample with absorbance value 0.077.
The graphic which based on theory is :

𝐴 𝐴

Dilution Concentratio
n

The graphic based on experiment result :

The Influence of Enzyme Dilution

to Enzyme Activity
0.45
0.4
0.35
Absorbance

0.3
0.25
0.2
0.15 Absorbance
0.1
0.05
0
0 20 40 60
Dilution

37 | P a g e
 The Influence of Enzyme Dilution
to Enzyme Activity
0.45
0.4
0.35
0.3
0.25
0.2
0.15 delta absorbance
0.1
0.05
0
0 10 20 30 40 50 60
Dilution

From the data, we can get the value of enzyme activity based on the
graphic. According to the theory, the graphic of enzyme activity can be
know by creating graphic dillution vs absorbance. Based on all the
result, we can conclude that the higher activity enzyme is happen at
10x dilution. Because at 10x dillution it has the smallest absorbance
value, which means that at 10x dilution absorption by the
spectrophotometer is getting smaller because many starches have been
hydrolyzed into glucose. And from the graphic dilution vs
absorbance we know how much amylum which has been
hydrolyzed, enzyme activity higher at 10x dilution it means that at that
point the amylum which hydrolyzed to become glucose has higher
value. This result is accordance with the theory which state that the
higher concentration enzyme will make the activity enzyme higher too.
J. CONCLUSION
1. Enzyme activity affected by pH, when the pH is too acid or too basic can
cause the loss of enzyme activity. According to the experiment result, pH
optimum of amylase enzyme of our sample is 9.0.
2. Enzyme activity affected by concentration, increasing the concentration of
enzyme also increasing the activity enzyme. Based on this experiment, the
10x dilution has lowest absorbance value which means that the less

38 | P a g e
 remaining value of amylum and the 50x dilution has highest absorbance
value.
3. REFERENSES
Akio, T., Masako MAEDA and Hidetoshi ARAKAWA. 1989.
Chemiluminescent Enzyme Immunoassay A Review. New York:
McGraw-Hill.
Beers RF, Jr., Sizer, I.W. 1951. A spectrophotometric method for measuring
the breakdown of hydrogen peroxide by catalase. J Biol Chem
1952;195:133–140, 1951.
Benkeblia, N. 2014. Polysaccharides: Natural Fibers in Food and Nutrition.
Boca Raton: CRC Press.
Boisseau, Patrick and Lahmani, Marcel. 2007. Nanoscience:
Nanobiotechnology and Nanobiology. London: Springer.
Butterly, John R., and Shepherd, Jack. 2010. Hunger: The Biology and Politics
of Starvation. Hanover: University Press of New England.
Campbell, Mary and Farrell, Shawn. 2009. Biochemistry. Belmont:
Brooks/Cole.
Chandrasekaran, M. 2015. Enzymes in Food and Beverage Processing. Boca
Raton: CRC Press.
Chesworth, J.M., Stuchburry, T., and Scaife, J. R. 2012. An Introduction to
Agricultural Biochemistry. London: Chapman & Hall.
Eaton, Louise and Rogers, Kara. 2018. Examining Basic chemical Molecules.
New York: Educational Publishing.
Emery, Alan E. H. 1968. Heredity, Disease, and Man. Los Angeles: University
of California Press.
Fischer, Johan. 2016. Islam, Standards, and Technoscience: In Global Halal
Zones. New York: Taylor and Francis.
Harisha, S. 2005. An Introduction to Practical Biotechnology. New Delhi:
Laxmi Publications.
Kokate , Chandrakant and Jalalpure, Pramod H.J, SS. 2011. Textbook of
Pharmaceutical Biotechnology. New Delhi: Elsevier.
Lehninger. 1982. Dasar-Dasar Biokimia. Jakarta: Erlangga.

39 | P a g e
Maclean, Norman. 2016. Dictionary of Genetics and Cell Biology. London:
Macmillan Press.
Macmillan, H. F. 1999. Tropical Planting and Gardening with Sepcial
Reference to Ceylon Fourth Edition. New Delhi: Asian Educational
Services.
Underwood, A.L., and Day, R.A. 1998. Analisis Kimia Kuantitatif. Jakarta:
Erlangga.
Pandey, A., Webb, C., Soccol, Carlos R., and Larroche, C. Enzyme
Technology. New Delhi: Springer.
Papachristodoulou, D., Snape, A., Elliott, William H., and Elliott, Daphne C.
2014. Biochemistry and Molecular Biology. Oxford: Oxford
University Press.
Patton, Kevin T., and Thibodeau, Gary A. 2017. The Human Body in Health &
Disease 7th Edition. St. Louis: Elsevier.
Piergiovanni, Angela R. and Herrero-Martínez, José Manuel. 2019. Analysis of
Peptides and Proteins by Electrophoretic Techniques. Basel: MDPI.
Poedjiadi, A. 1994. Dasar-Dasar Biokimia. Jakarta: Uiversitas Indonesia Press.
Pratama, A. P., Anggraeni, M., LFH, J. I., Amin, M., Amelia, R., & Jannah, A.
R. 2012. Pengaruh suhu dan pH terhadap aktivitas enzim. Jurnal
Kimia Indonesia, 1(1), 22-27.
Soewoto, H., et al. 2000. Biokimia Eksperimen Laboratorium. Jakarta: Widya
Medika.
Soni, N. K. 2010. Fundamentals Of Botany Volume 2. New Delhi: McGraw-
Hill.
Yamamoto, Takehiko. 1995. Enzyme Chemistry and Molecular Biology of
Amylases and Related Enzyme. Boca Raton: CRC Press

40 | P a g e
4. QUESTION ANSWER
1. Make a curve that shows the relation between the enzymatic reaction rate (∆A/
minumtes) with Ph!
pH Absorbance

Blank solution 1.875

1 1.256
3 0.363
5 0.769
7 0.820
9 1.648

The Influence of pH to Enzyme Activity

1.8
1.6
1.4
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒

1.2
1
0.8
delta absorbance
0.6
0.4
0.2
0
0 2 4 6 8 10
pH

2. Make a curve between enzyme dilution with enzymatic reaction rate (∆A/
minutes)!

Dilution Absorbance
Blank solution 0.460
10 0.383
20 0.363
30 0.171
40 0.055
50 0.083

41 | P a g e
 The Influence of Enzyme Dilution
to Enzyme Activity
0.45
0.4
0.35
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒

0.3
0.25
0.2
0.15 delta absorbance
0.1
0.05
0
0 10 20 30 40 50 60
Dilution

42 | P a g e
 ATTACHMENT

1. DOCUMENTATION

No Picture Description

Tools and Materials

1. Tools needed in this experiment

are: test tube, test tube rack,
volumetric flask 10 mL,
graduated cylinder 10 mL,
beaker glass 50 mL

2. Materials needed in this

experiment: Saliva, pH solution,
I2 solution, starch solution,
aquades

43 | P a g e
Enzyme Solution Dilution

1. Saliva was taken 1 mL for each

dilution

2. 1 mL saliva diluted with aquades

into 10 mL

3. Saliva with 10, 20, 30 ,40 and 50

times of dilution

Effect of pH on Enzyme Activity

44 | P a g e
1. 1 mL starch solution put into test
tube, added 0,5 mL aquades for
blank solution or 0,5 mL enzyme
of each dilution for test. Then it
left for 3 minutes in 37 C

2. Added 2 drops of iodine solution

3. Added 6 mL aquades

4. Measured the absorbance at 680

nm wavelength using
spectrophotometer

Effect of Concentration on Enzyme Activity

1. 1 mL of starch solution
measured with graduated
cylinder, then put into test tube

45 | P a g e
2. 1 mL of starch added 1 mL of
optimum pH in test tube

3. Added 0,5 mL aquades for blank

solution, and 0,5 mL enzyme in
each dilution for test, then left
for 3 minutes in 37 C

4. Let in > 60 C for 3 minutes

5. Added 2 drops of iodine solution

6. Added 6 mL of aquades

46 | P a g e
7. Read the absorbance using
spectrophotometer at 680 nm
wavelength

2. CALCULATION

pH Absorbance
1 0.619
3 1.512
5 1.106
7 1.055
9 0.227

The Influence of pH to Enzyme Activity

1.6
1.4
1.2
Absorbance

1
0.8
0.6 Absorbance
0.4
0.2
0
0 2 4 6 8 10
pH

pH Absorbance
Blank solution 1.875
1 1.256
3 0.363

47 | P a g e
 5 0.769
7 0.820
9 1.648

The Influence of pH to Enzyme Activity

1.8
1.6
1.4
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒

1.2
1
0.8
delta absorbance
0.6
0.4
0.2
0
0 2 4 6 8 10
pH

Dilution Absorbance
10 0.077
20 0.097
30 0.289
40 0.405
50 0.377

48 | P a g e
 The Influence of Enzyme Dilution
to Enzyme Activity
0.45
0.4
0.35
0.3
Absorbance

0.25
0.2
0.15 Absorbance
0.1
0.05
0
0 10 20 30 40 50 60
Dilution

Dilution Absorbance
Blank
solution 0.460
10 0.383
20 0.363
30 0.171
40 0.055
50 0.083

49 | P a g e
 The Influence of Enzyme Dilution
to Enzyme Activity
0.45
0.4
0.35
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒

0.3
0.25
0.2
0.15 delta absorbance
0.1
0.05
0
0 10 20 30 40 50 60
Dilution

50 | P a g e