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

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

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.

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

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

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

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

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.

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

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

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

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

groups

Disinfectant, kills

endospores

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

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.