This is aimed to explain the isolation of carbohydrates and starch from plant source. To also verify the presence of carbohydrates from the isolation process through several qualitative tests and qualitative tests for monosaccharides, disaccharides and polysaccharides

1. COMPILED
BY
OKANYA VIRGINUS.K.
ENZYME HYDROLYSIS OF STARCH
ABSTRACT
Enzymes are synthesized in a living cell and speed up reactions efficiently. It decreases
the energy of activation. The effectiveness of enzymes as catalysts is demonstrated by the very
high reaction rates at physiological temperatures. In the following experiments, the highly
specific nature of this is shown. The experiment is aimed to identify the factors affecting enzyme
catalysis and to demonstrate enzyme-catalyzed hydrolysis of starch using –amylase. The effect
of temperature and pH on the activity of salivary amylase on starch can be studied by using the
Iodine test. If we add saliva on starch, the salivary amylase present in saliva gradually acts on
starch and converts it into maltose. Starch keeps on giving blue colour with iodine till it is
completely digested into maltose. At this point, no blue colour is formed. This is the end point
or achromic point. The optimum temperature for the enzymatic activity of salivary amylase is 37
°C, the optimum pH for the enzymatic activity of salivary amylase is 7 while there was no
detectable disappearance of starch using disinfectant.
OBJECTIVE
1. To identify the factors affecting enzyme
catalysis.
2. To demonstrate enzyme-catalyzed
hydrolysis of starch using –amylase.
INTRODUCTION
Enzymes are synthesized in a living cell and
speed up reactions efficiently. It decreases
the energy of activation. The effectiveness
of enzymes as catalysts is demonstrated by
the very high reaction rates at physiological
temperatures. In the following experiments,
the highly specific nature of this is shown.
Enzymes are extracted from plants and

2. biological materials by different methods.
Amylase is an enzyme found in human
saliva and pancreas. It is the digestive
enzyme that is needed to break down starch
molecules. Amylase must be kept at certain
conditions to function at its optimum level.
This experiment will explore the effect of
PH, Temperature, and disinfectant on the
activity of amylase to hydrolyze the starch.
Starch is mainly composed of two
components, amylase (linear polymer of
glucose) which makes up 20-30% of the
structure; and amylopectin (branched
polymer of glucose).
Of the two components of starch,
amylopectin presents the great challenge to
hydrolytic enzyme systems. This is due to
the residues involved in a-1,6-glycosidic
branch points which constitute about 4 – 6%
of the glucose present. Most hydrolytic
enzymes are specific for a-1,4-glucosidic
links yet the a-1,6-glucosidic links must also
be cleaved for complete hydrolysis of
amylopectin to glucose.
MATERIALS
A. Equipment
(1) Triple beam balance Spot plate
(2) 100-mL beaker Hot plate
(1) Thermometer (6) 10mL Test tubes
B. Reagents
Lysol
NaHCO3
2% Starch PH 4 buffer solution
PH 10 buffer solution PH 7 buffer solution
(1) 25-mL graduated cylinder Amylase
0.2 M citric acid 0.2 KH2PO4
0.01 M NaOH Iodine solution
0.5% NaCl Starch solution
PROCEDURE
A. Preparation of a-Amylase from Saliva.
In a 25-mL graduated cylinder, 5-mL of
saliva was added, 15-mL of distilled water
and 5-mL of 0.5% NaCl were also added.
B. Preparation of starch solution

3. 1. 10 g of soluble corn starch was mixed in
50-mL of cold water.
2. While stirring, the slurry was added to
400-mL of gently boiling water in a large
beaker.
3. The gelatinized starch solution was well
mixed and cooled to room temperature.
4. More water was added to bring the total
volume to 500-mL.
5. A few drops of the starch solution was
placed on a glass plate. 1-drop of iodine
reagent was added and a deep was observed
developing.
C. Preparation of Buffers
PH 4: 0.2 M citric acid was adjusted to PH 4
with NaOH.
PH 7: 0.2 M monosodium dihydrogen
phosphate was adjusted to PH 7 with NaOH
PH 10: 0.2 M sodium bicarbonate was
adjusted to PH 10 with NaOH.
D. Effect of PH
1. Three test tubes were prepared and
labeled 1 – 3. To the first test tube 1-mL of
buffer (pH 4) was added; to the second, pH
7 buffer, and to the third , pH 10 buffer. To
each test tube 1 mL of 2% starch was added.
2. Another 3 test tubes were prepare and 2
mL of enzyme solution was placed to each
test tube. All the six tubes was incubated at
370C for 10 minutes.
3. Pairs of tubes were mixed and the
reaction at one was monitored at one minute
interval for 10 minutes by taking 3 drops
and it was transferred to a spot plate
containing iodine solution. The change in
color of the iodine was observed.
E. Effect of Temperature.
2 mL of enzyme solution were placed in
three separate test tubes and 2 mL of
buffered 1% starch (pH 7) solution in
another three separate test tubes. Pairs of
tubes were incubated for 10 minutes at
either 0, 37, or 700 C , then it was mixed and
processed as in procedure D(3). The change
in color in iodine solution was observed.
F. Effect of Disinfectant.
In one test tube, one drop of Lysol
disinfectant was added and 2 mL of enzyme
solution and at 370C it was pre-incubated
for 10 minutes along with 2 mL of 1% of
buffered starch in a separate tube. The two
test tubes were mixed immediately and
processed as in procedure D(3). The change
in color in iodine solution was observed.

4. RESULTS
Effect of pH
p
H
Change in color of iodine at different time interval (minutes)
1 2 3 4 5 6 7 8 9 10
4 Brown Brown Brown Brown Dark
Brown
Dark
Brown
Dark
Brown
Dark
brown
Black Black
7 Slightly
blue
Slightly
blue
Light
Blue
Light
blue
Blue Blue Deep
Blue
Deep
Blue
Brow
nish
Blue
BRo
wn
10 Black Black Black Black Black Black Black Black Black Black
A brown color was recorded for the first 4 minutes, dark brown for the 5 – 7th minutes, dark
color for 9 and 10th minutes for the pH 4 test. For pH 7 slightly blue color for the first 2 minutes,
light blue for 3 and 4th minutes, blue color for 5 and 6th minutes, deep blue for 7 and 8th minutes,
brownish blue for 9th and Brown for 10th minutes respectively. While pH 10 showed black color
all through.
Ph 4 pH 7 pH 10

5. Effect of Temperature
Temperat
ure (0C)
Change in color of iodine at different time interval (minute)
1 2 3 4 5 6 7 8 9 10
0 Brown Brown Brow
n
Brow
n
Dark
Brow
n
Dark
Brow
n
Dark
Brow
n
Dark
Brow
n
Black Black
37 Slightl
y Blue
Slightly
Blue
Light
Blue
Light
Blue
Blue Blue Deep
Blue
Deep
Blue
Brow
n
Brow
n
70 Black Black Black Black Black Black Black Black Black Black
For the effect of temperature, 00C recorded brown for the first 4 minutes, dark brown for 5 – 8th
minutes, and black for 9 and 10th minutes. For 370 C, slightly blue color for the first 2 minutes,
light blue for 3 and 4th minutes, blue color for 5 and 6th minutes, deep blue for 7 and 8th minutes,
brownish blue for 9th and Brown for 10th minutes respectively. While 700 C also showed black
color all through.
Effect of Disinfectant

6. Disinfecta
nt
Change in color of iodine at different time interval (minute)
1 2 3 4 5 6 7 8 9 10
Lysol Slig
htly
Blue
Slig
htly
Blu
e
Brown Brow
n
Brow
n
Brow
n
Brow
n
Brow
n
Brow
n
Blac
k
For the effect of Disinfectant, Slightly Blue color was observed for first 2 minutes, Brown color
from 3 – 9th minutes, and the 10th minute is black
DISCUSSION
All living beings need energy to
survive. It is from the food we consume that
we get our energy. We know that the energy
we are getting is by the process of digestion
that breaks down the complex substance of
starch into simpler molecules of glucose,
which are further metabolized into CO2 and
water through the process of glycolysis. The
human digestive tract starts at the mouth and
ends at the anus. The digestion of the food
starts as soon as we put food in our mouth.
Our teeth cut the food into small pieces and
the salivary glands secrete saliva that mixes
with these food materials. The saliva
contains an enzyme called salivary amylase

7. which hydrolyses starch into maltose. The
complete digestion of starch occurs only in
the small intestine by the action of
pancreatic amylase.
The effect of temperature, pH and
disinfectant on the activity of salivary
amylase on starch was studied by using the
Iodine test. When saliva added on starch,
the salivary amylase present in saliva
gradually acts on starch and converts it into
maltose. Starch keeps on giving blue colour
with iodine till it is completely digested into
maltose. At this point, no blue colour is
formed which is the end point or achromic
point.
Effect of pH
From the above results obtained on the
effect of pH for the enzymatic activity of
salivary amylase, we observed that the
optimum pH on the enzymatic activity of
salivary amylase is pH 7. This is because
there is a theoretical prove that the positive
test of salivary amylase on iodine is blue and
only pH 7 showed positive test to that.
Above and below this range, that pH 10 and
pH 4 the reaction rate reduces as enzymes
get denaturated. The enzyme salivary
amylase is most active at pH 6.8. Our
stomach has high level of acidity which
causes the salivary amylase to denature and
change its shape. So the salivary amylase
does not function once it enters the stomach
Effect of Temperature
All enzymes are proteinaceous in nature. At
a lower temperature, the enzyme salivary
amylase is deactivated and at the higher
temperature, the enzyme is denaturated.
Therefore, more time will be taken by an
enzyme to digest the starch at lower and
higher temperatures. From the above results
obtained on the effect of temperature on the
enzymatic activity of salivary amylase, we
observed that the optimum temperature for
the enzymatic activity of salivary amylase is
370C. This is also because there is a
Stheoretical prove that the positive test of
salivary amylase on iodine is blue and only
at 370C temperature showed positive test to
that Optimum temperature for the
enzymatic activity of salivary amylase
ranges from 32 °C to 37 °C. The optimum
temperature means that the temperature at

8. which the enzyme shows the maximum
activity. At this optimum temperature, the
enzyme is most active and hence, takes less
time to digest the starch.
Effect of Disinfectant
Generally, there was no detectable
disappearance of starch in this test. Lysol at
this strength seems to interfere with color
stability, causing the blue color to last only
briefly, so we tried to record observations
immediately after mixing. (Diluted Lysol
solutions are less effective at inhibiting the
enzyme.
CONCLUSION
The process of digestion of starch by
salivary amylase was well studied and the
effect of temperature, pH and Disinfectant
on the activity of salivary amylase on starch
was well understood. The optimum
temperature for the enzymatic activity of
salivary amylase is 37 °C, the optimum pH
for the enzymatic activity of salivary
amylase is 7 while there was no detectable
disappearance of starch using disinfectant.
Above and below this range, the reaction
rate reduces as enzymes get denaturated.
The above analyses is concluded to be right
due to the color changes at different
temperature and pH, meanwhile 370C
temperature and the pH7 have the same
color as the known reference for Amylase
and iodine test.
ANSWERS TO THE QUESTION
1. Are all enzymes in nature work best at pH 7 and 370C? Explain.
The activity of enzymes is affected by temperature and pH but the answer is Yes for
Temperature and No for pH because;
Temperature and enzymes
As the temperature increases, so does the rate of reaction. But very high temperatures
denature enzymes.

9. The graph shows the typical change in an enzyme's activity with increasing temperature. The
enzyme activity gradually increases with temperature up to around 37ºC, or body temperature.
Then, as the temperature continues to rise, the rate of reaction falls rapidly as heat energy
denatures the enzyme.
Graph of enzyme activity against temperature
pH and enzymes
Changes in pH also alter an enzyme’s shape. Different enzymes work best at different pH values.
The optimum pH for an enzyme depends on where it normally works. For example, intestinal
enzymes have an optimum pH of about 7.5. Enzymes in the stomach have an optimum pH of
about 2.
Graph of enzyme activity against pH

10. 2. Explain why heat, cold and disinfectant prevents microbial growth,
Applications of Heat
The lethal temperature varies in microorganisms. The time required to kill depends on the
number of organisms, species, nature of the product being heated, pH, and temperature.
Autoclaving, which kills all microorganisms with heat, is commonly employed in canning,
bottling, and other sterile packaging procedures. This is an ultimate form of preservation against
microbes. But, there are some other uses of heat to control growth of microbes although it may
not kill all organisms present. Boiling: 100o for 30 minutes (more time at high altitude). Kills
everything except some endospores. It also inactivates viruses. For the purposes of purifying
drinking water, 100o for five minutes is a "standard" in the mountains" though there have been
some reports that Giardia cysts can survive this process.
Recommended use of heat to control bacterial growth
Treatment Temperature Effectiveness
Incineration >500o
Vaporizes organic material on
nonflammable surfaces but
may destroy many substances
in the process
Boiling 100o
30 minutes of boiling kills
microbial pathogens and
vegetative forms of bacteria
but may not kill bacterial
endospores
Intermittent boiling 100o
Three 30-minute intervals of
boiling, followed by periods of

11. cooling kills bacterial
endospores
Autoclave and
pressure cooker (steam
under pressure)
121o/15
minutes at 15#
pressure
kills all forms of life including
bacterial endospores. The
substance being sterilized
must be maintained at the
effective T for the full time
Dry heat (hot air oven) 160o/2 hours
For materials that must remain
dry and which are not
destroyed at T between
121o and 170o Good for
glassware, metal, not plastic or
rubber items
Dry heat (hot air
oven)
170o/1 hour
Same as above. Note
increasing T by 10 degrees
shortens the sterilizing time by
50 percent
Pasteurization (batch
method)
63o/30
minutes
kills most vegetative bacterial
cells including pathogens such
as streptococci, staphylococci
and Mycobacterium
tuberculosis
Pasteurization (flash
method)
72o/15
seconds
Effect on bacterial cells
similar to batch method; for
milk, this method is more
conducive to industry and has
fewer undesirable effects on
quality or taste
Ultrapasteurization
(direct method)
140o/2
seconds
Effect on most bacterial cells
is lethal. For milk, this method

12. creates a product with
relatively long shelf life at
refrigeration temperatures.
APPLICATION OF COLD
Most organisms grow very little or not at all at 0oC. Perishable foods are stored at low
temperatues to slow rate of growth and consequent spoilage (e.g. milk). Low temperatures are
not bactericidal. Psychrotrophs, rather than true psychrophiles, are the usual cause of food
spoilage in refrigerated foods. Although a few microbes will grow in supercooled solutions as
low as minus 20oC, most foods are preserved against microbial growth in the household freezer.
APPLICATION OF DISINFECTANT
Antimicrobial agents are chemicals that kill or inhibit the growth microorganisms. Antimicrobial
agents include chemical preservatives and antiseptics, as well as drugs used in the treatment of
infectious diseases of plants and animals. Antimicrobial agents may be of natural or synthetic
origin, and they may have a static or cidal effect on microorganisms.
Common antiseptics and disinfectants
Chemical Action Uses
Ethanol (50-70%)
Denatures proteins
and solubilizes
lipids
Antiseptic used on skin
Isopropanol (50-70%)
Denatures proteins
and solubilizes
lipids
Antiseptic used on skin
Formaldehyde (8%)
Reacts with NH2,
SH and COOH
Disinfectant, kills
endospores

13. groups
Tincture of Iodine (2% I2
in 70% alcohol)
Inactivates proteins
Antiseptic used on skin
Disinfection of
drinking water
Chlorine (Cl2) gas
Forms
hypochlorous acid
(HClO), a strong
oxidizing agent
Disinfect drinking
water; general
disinfectant
Silver nitrate (AgNO3)
Precipitates
proteins
General antiseptic and
used in the eyes of
newborns
Mercuric chloride
Inactivates proteins
by reacting with
sulfide groups
Disinfectant, although
occasionally used as an
antiseptic on skin
Detergents (e.g.
quaternary ammonium
compounds)
Disrupts cell
membranes
Skin antiseptics and
disinfectants
Phenolic compounds (e.g.
carbolic acid, lysol,
hexylresorcinol,
hexachlorophene)
Denature proteins
and disrupt cell
membranes
Antiseptics at low
concentrations;
disinfectants at high
concentrations
Ethylene oxide gas Alkylating agent
Disinfectant used to
sterilize heat-sensitive
objects such as rubber
and plastics
Ozone Generates lethal
oxygen radicals
Purification of water,
sewage

14. 3. What are the optimum conditions of a-amylase enzyme as regard to temperature and pH.
TEMPERATURE
There are two temperatures that need to be in optimum range during production. They are
temperature for the growth of the microbial source and optimum temperature at which maximum
production of enzyme takes place. The optimum temperatures for growth and α-Amylase
production were found to be 45°C to 46 °C and 50 °C, respectively.
pH
Optimum pH is a critical factor for the stability of enzyme produced. Enzymes are pH sensitive
and hence care must be taken to control the pH of the production process. The study revealed
that the amylase exhibited maximal activity at pH 7.0, being relatively stable in alkaline
conditions.