Testing the activity of immobilized enzymes by using chemical tests

1. Testing of Immobilized
enzyme beads
Presented by
Pramod Channammanavar
Bpharm 7th semester
Bapuji Pharmacy College, Davangere

2. CONTENTS
 Definitions of
 Immobilization
 Immobilized enzyme
 Testing the activity of Immobilized enzyme
 Requirements
 Principle
 Procedure
 Conclusion/Report
 References

3. What is Enzyme immobilization?
Enzyme immobilization can be defined as the confinement of
enzyme molecules within a matrix (physically or chemically or
both),
in such a way that it retains its full activity or most of its activity.
What is an Immobilized enzyme?
An Immobilized enzyme is one whose movement
has been restricted either completely or to a
small limited region

4. Testing of Immobilized enzyme
beads
• Requirements: Test tubes, Std Maltose solution, DNSA
reagent (3,5-Dinitro salicylic acid),1% starch in acetate
buffer of pH 4.8, Acetate buffer
• Principle: Usually immobilization leads to the slight
reduction in the activity of enzyme. Hence, it is necessary
to determine its activity. Β-amylase catalyses the
hydrolysis of α-1,4 linkages of starch with formation of
maltose. Maltose though disaccharide it is reducing sugar.
The enzyme reaction can be stopped by DNS reagent. DNS
is reduced to 3-Amino-5-nitro salicylic acid. This reaction
of DNS can be identified by the Change of color from
yellow to red.

5. Procedure
• Take 4 test tubes, the reagents are added into the test tube as shown in the
table.
Sl
number
Name Starch Amount
of
solutions
Amount
of DNSA
01 Positive
control
0.5 ml 0.5 ml of
Maltose
1 ml
02 Native
control
0.5 ml 0.5 ml of
Native
enzymes
1 ml
03 Immobili
zed
enzyme
(Beads)
0.5 ml 4-6
Beads
1 ml
04 Negative
control
0.5 ml 0.5 ml of
Water
1 ml
Incubate
at room
tempera
ture for
5
minutes
___________________________________________________________

6. Procedure
• After addition of DNSA the test tubes are
heated in hot water bath for 5 minutes. Till
the color change

7. Procedure
• The test tubes are taken change in the color is
observed.

8. Conclusion
Among 4 test tubes
1. Postive control group shows shows Red colour
2. Native control group also shows Red colour
3. Immobilized enzyme beads shows Yellow colour
4. Negative control group also shows Yellow colour

9. Conclusion
• Change in colour from yellow to Reddish
colour indicates enzymatic activity,
• Where as Yellow colour (No change in
colour from yellow to red) indicates no
enzymatic activity

10. REFERENCES
• Methods in Enzymology
Volume 571, 2016
• Internet source( https://youtu.be/9znHZ7VAXBU)
• https://en.m.wikipedia.org/wiki/Enzyme_assayymologyVo
lume
• And Information is also collected from the experiment
that we performed on 28/01/2022 under the guidance of
PGS Mam