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Experiment 4 - Effect of Enzyme Concentration On Enzyme Activity

This experiment aims to determine the effect of enzyme concentration on the activity of salivary amylase. Saliva contains the enzyme amylase, which catalyzes the hydrolysis of starch into maltose. Test tubes containing varying concentrations of saliva enzyme were incubated with starch solution, and enzyme activity was measured using a colorimetric assay. The results showed that amylase activity increased with increasing enzyme concentration up to an optimal level, beyond which further increases in concentration did not further increase activity. The optimum enzyme concentration for salivary amylase under these experimental conditions was determined to be ____. A second part of the experiment examined the effect of temperature on amylase activity and found that activity decreased with increasing temperature

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602 views

Experiment 4 - Effect of Enzyme Concentration On Enzyme Activity

This experiment aims to determine the effect of enzyme concentration on the activity of salivary amylase. Saliva contains the enzyme amylase, which catalyzes the hydrolysis of starch into maltose. Test tubes containing varying concentrations of saliva enzyme were incubated with starch solution, and enzyme activity was measured using a colorimetric assay. The results showed that amylase activity increased with increasing enzyme concentration up to an optimal level, beyond which further increases in concentration did not further increase activity. The optimum enzyme concentration for salivary amylase under these experimental conditions was determined to be ____. A second part of the experiment examined the effect of temperature on amylase activity and found that activity decreased with increasing temperature

Uploaded by

LinhNguye
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© © All Rights Reserved
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EXPERIMENT 4 – EFFECT OF ENZYME

CONCENTRATION ON ENZYME ACTIVITY

Objectives

To determine the effect of enzyme concentration on the activity of salivary amylase.

Background
Amylase, an enzyme present in saliva, catalyzes the hydrolysis of the glycosidic
linkages in starch. The effect of enzyme concentration, substrate concentration, temperature,
pH, and heavy metal cations on the activity of this enzyme will be investigated.
Metabolism
The chemical reactions that go on in living organisms, and which are essential for
growth, reproduction, movement, and all other vital functions, are individually and
collectively referred to as metabolism. Many thousands of such metabolic processes are
occurring constantly in even the simplest forms of life.
Enzymes are proteins that act as catalysts for metabolic reactions. They increase the rate
of the reaction, but do not influence the kind or amount of products formed. In general, each
metabolic reaction has to be catalyzed in the living organism by its own special enzyme.
The existence of enzymes in biological materials can be demonstrated by the effects they
bring about. In this experiment, digestive enzymes capable of breaking down such
polysaccharides as starch will be studied.
Food materials must be broken down by digestion before they can be absorbed and
utilized by the body. Consequently, if an organism has no enzyme capable of attacking a
particular type of carbohydrate, protein, or fat, that particular substance will have no food
value for the organism. Carbohydrates and fats can be broken down by strong acids or bases.
However, digestive enzymes can accomplish the same breakdown under conditions
compatible with life, that is, at moderate temperatures and physiological pH ranges.
Starch
The nutritional reservoir in plants is starch, which is actually a mixture of two
polysaccharides. Amylose, the unbranched type of starch, consists of glucose residues in α-l,4
linkage.

Amylopectin, the branched form, has about one α -l,6 1inkage per thirty α -l,41inkages.
Amylases
Hydrolysis of starch is the process of digestion. Enzymes called amylases catalyze only
the hydrolysis of α -1,4 glycosidic linkages in the amylose and amylopectin components of
starch. The hydrolysis does not proceed directly from polysaccharides to monomer units;
rather, partial hydrolysis products of intermediate size are obtained. These products are
maltose and dextrin. Maltose consists of two glucose units in α -1,4 linkage; dextrin is made
up of several glucose units joined by α -1,6 linkage in addition to α -1,4 linkages.
Amylase enzymes are present in saliva, so the digestion of carbohydrates begins in the
mouth. Digestion continues briefly in the stomach until the pH drops too low, and then is
completed in the intestines by the attack of another amylase.
The chemical conversion of starch to dextrins and maltose is a key part of the brewing
step in the production of beer. The amylase enzymes are added to the grain mixture as
brewer's malt. Approximately one third of the calories in regular beer are contributed by
carbohydrate remaining in the beer after fermentation. The carbohydrates remain because
amylase is unable to break down the α -l,6 glycosidic linkages in the amylopectin portion of
the starch. To make "light beer," less starch is used in the initial brewing step and a second
enzyme, amyloglucosidase, which can cleave the α -1,6 glycosidic linkages in amylopectin, is
added in the fermentation step. The light beer will have a lower carbohydrate composition
because of the added amyloglucosidase, and a lower alcohol content because of the reduced
starch in the initial fermentation mixture.

Principle
Amylase present in salivary secretion hydrolysis α1,4-linked D-Glucose units and
produce maltose as a final product.

Materials
1. Substrate : 1% Starch solution is boiled with phosphate buffer
2. Activator : 1% Sodium chloride
3. Inhibitor : 2N Sodium hydroxide
4. Buffer : Phosphate buffer (pH 6.8)
Solution A : 0.2m Disodium hydrogen phosphate-28.392g/1000ml distilled water.
Solution B : 0.2m Sodium dihydrogen phosphate-31.202g/1000ml distilled water.

S.No Solution A(ml) Solution B(ml) pH

1. 49.0 51.0 6.8

5. Dinitro salicylate: 30g of sodium potassium tatrate, 1g of 3,5- Dinitro salicylate and
1.6g of sodium hydrozide is weighed and made up to 100 mL

6. Enzyme source : 1ml of saliva is diluted to 20 mL.

Procedure (Strictly follow the provided table)


1. To a set of test tubes marked as ET and EB pipette out 2.5ml of buffer (pH 6.8 ) solution.
2. Add 2.5ml of substrate starch followed by 1ml of activator to all the tubes.
3. Now in the tubes marked as ET add 0.2ml -1.0ml of enzyme is added and are incubated at
37’ C for 15min.
4. The reaction is arrested by adding 0.5ml sodium hydroxide.
5. Add 0.2ml -1.0ml of enzyme in the tubes marked as EB and all the tubes are made upto
7ml using distilled water.
6. Add 0.5ml of DNS to all the tubes, then the tubes are boiled for 5’min. The colour
developed read at 540nm.

No. Contents B EB1 ET1 EB2 ET2 EB3 ET3 EB4 ET4 EB5 ET5

Volume of Buffer
1 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5
(mL)
Volume of
2 - 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5
Substrate (mL)
Volume of
3 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Activator (mL)
Volume of Enzyme
4 - - 0.2 - 0.4 - 0.6 - 0.8 - 1.0
(mL)

Incubate all the tubes at 37 ˚C for 10 min

Volume of distilled
5 1.0 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 - -
water (mL)
Volume of NaOH
6 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
(mL)
Volume of Enzyme
7 - 0.2 - 0.4 - 0.6 - 0.8 - 1.0 -
(mL)
Volume of DNS
8 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
(mL)

Keep all the tubes in boiling water bath for 5 min

Optical density at
9
540 nm
10 Difference in OD

RESULT
Optimum enzyme concentration for the enzyme salivary amylase is _____________
EXPERIMENT 5 – EFFECT OF HEAT STABILITY ON
ENZYME ACTIVITY
Objectives

To determine the heat stability of salivary amylase.

Materials
1. Substrate : 1% Starch solution
2. Activator : 1% Sodium chloride
3. Inhibitor : 2N Sodium hydroxide
4. Buffer : Phosphate buffer
Solution
DisodiumA hydrogen
: 0.2m phosphate-28.392g/1000ml distilled water.
Solution
Sodium dihydrogen
B : 0.2mphosphate-31.202g/1000ml distilled water.

S.No Solution A(ml) Solution B(ml) pH

1. 49.0 51.0 6.8

5. Dinitro salicylate:30g of sodium potassium tatrate, 1g of 3,5- Dinitro salicylate and 1.6g of
sodium hydroxide is weighed and made upto 100ml
6. Enzyme source :1ml of saliva is diluted to 20ml.

Procedure

1. A set of test tubes marked as ET and EB are taken.


2. Add 2.5ml of buffer (6.8 PH ), 2.5ml of substrate followed by 1ml of activator to all the
tubes, the tubes are then preincubated at 37’ C for 10min.
3. Add 0.5ml of enzyme to tubes marked as ET and incubate tubes at the respective
temperature for 15min
4. The reaction is arrested by adding 0.5ml of sodium hydroxide
5. Add 0.5ml of enzyme to the test tube marked as EB
6. 0.5ml of DNS is added to all the test tubes
7. The tubes are boiled for 5’min.
8. The colour developed is read at 540nm
40˚C 50˚C 60˚C 70˚C 80˚C
No. Contents B
EB1 ET1 EB2 ET2 EB3 ET3 EB4 ET4 EB5 ET5
Volume of
1 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5
Buffer (mL)
Volume of
2 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5
Substrate (mL)
Volume of
3 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Activator (mL)
Preincubate all the tubes at 37 ˚C for 10 min
Volume of
4 distilled water 0.5 - - - - - - - - - -
(mL)
Volume of
5 - - 0.5 - 0.5 - 0.5 - 0.5 - 0.5
enzyme (mL)
Incubate tubes at the respective temperature for 15 min
Volume of
6 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
NaOH (mL)
Volume of
7 - 0.5 - 0.5 - 0.5 - 0.5 - 0.5 -
enzyme (mL)
Volume of
8 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
DNS (mL)
Keep all the tubes in boiling water bath for 5 min
Optical density
9
at 540 nm
Difference in
10
OD

Result

Salivary amylase has its heat stability up to ____________

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