Introduction Why immobilize enzymes? Methods for immobilization of enzymes. Physical methods :- Adsorption, Entrapment, Encapsulation, Chemical methods:-, Covalent binding , Cross linking, Uses of Immobilized enzymes. Limitations Conclusions References

1. IMMOBILIZATION OF ENZYMES
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. SYNOPSIS
Introduction
Why immobilize enzymes?
Methods for immobilization of enzymes.
Physical methods :-
Adsorption
Entrapment
Encapsulation
Chemical methods:-
Covalent binding
Cross linking
Uses of Immobilized enzymes.
Limitations
Conclusions
References

3. INTRODUCTION
What is immobilization?
The covalent attachment of an enzyme to a solid matrix
(like sepharose) so that it cannot move but still act on its
substrate.
An enzyme can be linked to an inert support material
without loss of activity which facilitates reuse and recycling of
the enzyme. Enzymes can also be encapsulated or entrapped.
IMMOBILIZED ENZYME
Enzymes or cells which are physically confined to a
defined region in space while retaining their catalytic activity
and have the ability to be repeatedly and continuously used.

4. WHY IMMOBILIZE ENZYMES?
Protection from degradation and deactivation.
Re-use of enzymes for many reaction cycles, lowering the
total production cost of enzyme mediated reactions.
Ability to stop the reaction rapidly by removing the enzyme
from the reaction solution.
Enhanced stability.
Efficiency of immobilized enzyme is better and therefore
high product yield.
Easy separation of the enzyme from the product.
Suitable for industrial and medical use.
Product is not contaminated with the enzyme.

5. 3 MAJOR CONSTITUENTS FOR ENZYME
IMMOBILIZATION
SOLUBLE ENZYME – Isolated by fermentation or from
animal tissues or plant cells.
Should be stable
High specific activity.
Desirable operational parameters.
CARRIER – Should be insoluble in reaction environment.
Non-toxic.
Resistance to microbial, thermal or mechanical degradation.
Large no of sites for binding of enzymes.
Regenerability and cost.
METHODS OF IMMOBILIZATION
SOURCE
PURITY

6. CLASSIFICATION OF CARRIERS
Inorganic
Carriers
•High pressure
stability.
• May undergo
abrasion
Examples:
1. Commercialy SiO2
available
materials-
o Porous glass.
o Silica.
2. Mineral materials -
(clays)
Celite ,Centonite
Organic Natural
Carriers
•Favourable
compatibility with
proteins.
Examples:
1. cellulose
derivatives-
o DEAE-cellulose
o CM-cellulose.
2. Dextran.
3. Polysacharides
Agarose, Starch
Pectine ,Chitosan
Organic Synthetic
Carriers
•High chemical
and mechanical
stability.
Examples:
1. Polystyrene
2.Polyvinylacetate
3. Acrylic
polymers

7. Methods for enzyme
immobilization
PHYSICAL CHEMICAL
ADSORPTION
ENTRAPMENT
ENCAPSULATION
COVALENT BINDING
SUPPORT CROSS
LINKING

8. PHYSICAL METHODS FOR
IMMOBILIZATION
ADSORPTION
 Involves the physical binding of the enzyme on the
surface of carrier matrix.
 Carrier may be organic or inorganic.
 The process of adsorption involves the weak interactions
like vander waal or hydrogen bonds.
Carriers: - silica, bentonite, cellulose, etc.
e.g. catalase & invertase

9. ADVANTAGES
1. Simple and economical
2. Limited loss of activity
3. Can be Recycled, Regenerated & Reused.
DISADVANTAGES
1. Relatively low surface area for binding.
2. Exposure of enzyme to microbial attack.
3. Yield are often low due to inactivation and
desorption.

10. ENTRAPMENT
1. In entrapment, the enzymes or cells are not directly
attached to the support surface, but simply trapped inside
the polymer matrix.
2. Enzymes are held or entrapped within the suitable
gels or fibres.
3. In gel it may causes :
 Matrix polymerization or
 Precipitation or
 Coagulation
4. Entrapment in calcium alginate is the most widely
used for entrapment for:-
 Animal and plant cells enzymes.

12. Contd..
It is done in such a way as to retain protein while allowing
penetration of substrate. It can be classified into lattice and
micro capsule types.
A. Inclusion in gels: Poly acrylamide gel, Poly vinyl alcohol
gels.
B. Inclusion in fibers: Cellulose and Poly - acrylamide gels.
C. Inclusion in micro capsules: Polyamine, Polybasic acid
chloride monomers.

13. Lattice-Type Entrapment
Entrapment involves entrapping enzymes within the
interstitial spaces of a cross-linked water-insoluble
polymer. Some synthetic polymers such as polyarylamide,
polyvinylalcohol, etc... and natural polymer (starch) have
been used to immobilize enzymes using this technique.

14. MicrocapsuleType Entrapmet/
Encapsulation/Membrane Confinement
It involves enclosing the enzymes within semi -
permeable polymer membranes e.g. semipermeable
collodion or nylon membranes in the shape of spheres
are utilized for microencapsulation of Aminocyclase
enzyme.
Encapsulation of enzyme

15. PROCEDURES FOR
MICROENCAPSULATION OF ENZYMES
INTERFACIAL POLYMERIZATION METHOD
LIQUID DRYING
PHASE SEPARATION

16. INTERFACIAL POLYMERIZATION METHOD
HYDROPHILIC MONOMER + ENZYME (ASPARAGINASE)
(NH2(CH2)6 NH2)
BUFFERED
EMULSIFIED IN WATER IMMISCIBLE ORGANIC
SOLVENT ( CYCLOHEXANE+CHLOROFORM)
HYDROPHOBIC MONOMER
(SEBACOYL CHLORIDE)
POLYMERIZATION OF THE MONOMERS OCCURS AT THE
INTERFACE BETWEEN THE AQUEOUS AND ORGANIC SOLVENT
PHASES.
REACTION GIVES THE
NYLON POLYMER

17. ADVANTAGES
1. No chemical modification.
2. Relatively stable forms.
3. Easy handling and reusage.
DISADVANTAGES
1. The enzyme may leak from the pores.

18. CROSS LINKING
Cross linking involves intermolecular cross linking of
enzyme molecules in the presence/absence of solid support.
The method produces a 3 dimensional cross linked
enzyme aggregate (insoluble in water) by means of a
multifunctional reagent that links covalently to the enzyme
molecules.
Cross linking involves several multifunctional reagents :-
Diazobenzidine
Glutaraldehyde
1,5 difluro 2,4 dinitrobenzene
Hexamethylene di-isocyanate

20. Advantages of cross linking:-
1. Very little desorption(enzyme strongly bound)
2. Higher stability (i.e. ph,ionic & substrate concentration)
Disadvantages of cross linking:-
1. Cross linking may cause significant changes in the active
site.
2. Not cost effective.

21. COVALENT BINDING
METHOD
1. Based upon the formation of covalent bonds between
enzymes and the support matrix under mild conditions.
2. The covalent linkage is accomplished through
functional groups that are not involved in catalytic function.
3. Some examples of covalent linkage between the
support and the enzyme are as follows :-
Support with –OH group

22. CONTD…..
Support having –NH2 group
Support with -COOH group

23. SUPPORT WITH –OH GROUP INVOLVING CNBr ACTIVATION

24. Advantages of covalent coupling :-
1. The strength of binding is very strong, so, leakage of
enzyme from the support is absent or very little.
2. This is a simple, mild and often successful method of
wide applicability
Disadvantages of covalent coupling :-
1. Enzymes are chemically modified and so many are
denatured during immobilization.
2. Only small amounts of enzymes may be immobilized
(about 0.02 grams per gram of matrix).

26. 1. ENZYMES IN WASHING POWDER
PROTEASES are used to digest away proteins present in
blood stains, milk and also in association with dirt. Only
serine proteases are suitable for use in detergents.
CELLULASES , produced by fungi are used for washing cotton
fabrics.
2. PRODUCTION OF HIGH FRUCTOSE SYRUP
3. IMMOBILIZED ENZYMES ARE WIDELY USED IN
BIOSENSORS.
APPLICATIONS

27. DIAGRAMATIC REPRESENTATION OF
A BIOSENSOR

28. APPLICATIONS
4. IMMOBILIZES ENZYMES USED IN FOOD INDUSTRY
A. Pectinase break down substances in apple cell walls and
enable greater juice extraction.
B. Lactase breaks down lactose in milk into glucose and
galactose. This makes milk drinkable for lactose
intolerant people.
5. DIAGNOSIS OF MEDICAL CONDITION
A. Prostate cancer is detected using immobilized acid
phosphatise.
B. Immobilized enzymes like phosphatise and beta
lactamase are used in analysis of antigen and antibodies.

29. DISADVANTAGES
1. Immobilization means additional cost. Therefore it
should be used only when there is a sound economic,
safety or process advantages over soluble enzymes.
2. Immobilization often adversely affects the stability
and/or activity of the enzymes, In such cases, suitable
immobilization protocols should be developed.
3. Immobilized cells are particularly vulnerable to
contamination. An antibiotic may have to be used in
such cases.

30. CONCLUSIONS
Enzyme immobilization is one of the most promising
approaches for exploiting enzyme based processes in
Biotransformation,diagnostics,pharmaceutical and food
industries. Several hundreds of enzymes have been
immobilized in a variety of forms including penicillin G
Acylase, lipases, proteases, invertase, etc. And are being
currently used as catalysts in various large scale
processes.

31. REFERENCES
 http://www.scribd.com/doc/31429014/Immobilized-Enzymes
 http://enzymetechnology.blogspot.in/2009/10/enzyme-technology.html
 http://www.biotecharticles.com/Biotech-Research-Article/Enzyme-Immobilization-
Methods-and-Applications-460.html
 http://enzymeimmobilization.blogspot.in/2011/02/enzyme-immobilization.html