Dr.S.Karthikumar Asst. Prof., Dept. of Biotechnology Kamaraj College of Engineering and Technology S.P.G.C.Nagar, Virudhunagar, Tamilnadu, India skarthikumar@gmail.com

1. Dr.S.Karthikumar

2. What Is An Immobilized Enzyme?
An immobilized enzyme is one whose
movement in space has been restricted either
completely or to a small limited region.
Enzyme immobilization may be defined as a
process of confining the enzyme molecules
to a solid support over which a substrate is
passed and converted to products.
What Is Enzyme Immobilization ?

3. 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.
 Easy separation of the enzyme from the product.
 Product is not contaminated with the enzyme.

4. An Ideal Carrier Matrices For Enzyme
Immobilization
 Inert.
 Physically strong and stable.
 Cost effective.
 Regenerable.
 No product inhibition.

5. CLASSIFICATION OF CARRIERS
Inorganic
Carriers
•High pressure
stability.
• May undergo
abrasion
Examples:
o Porous glass.
o Silica.
o Mineral materials -
(clays)
Celite, Bentonite
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

6. METHODS FOR ENZYME
IMMOBILIZATION
PHYSICAL CHEMICAL
ADSORPTION
ENTRAPMENT
ENCAPSULATION
COVALENT BINDING
SUPPORT CROSS
LINKING

7. 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

8. Methods of adsorption:
 (1). Static process: Immobilization to carrier by allowing the solution
containing enzyme to contact the carrier without stirring.
(2). Dynamic batch process: Carrier is placed in the enzyme solution and
mixed by stirring or agitation.
(3). Reactor loading process: Carrier is placed in the reactor, and then the
enzyme solution is transferred to the reactor with continuous agitation.
(4). Electrode position process: Carrier is placed near to an electrode in an
enzyme bath and then the current is put on, under the electric field the enzyme
migrates to the carrier and deposited on its surface.

9. ADVANTAGES DISADVANTAGES
 1. Simple and economical
 2. Limited loss of activity
 3. Can be Recycled,
Regenerated & Reused.
 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
 In entrapment, the enzymes or cells are not directly attached to the support
surface, but simply trapped inside the polymer matrix.
 Enzymes are held or entrapped within the suitable gels or fibres.
 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.
Inclusion in gels: Poly acrylamide gel, Poly vinyl alcohol gels.
Inclusion in fibers: Cellulose and Poly -acryl amide gels.
Inclusion in micro capsules: Polyamine, Polybasic -
acid chloride monomers.

11. 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.

12.  Advantages of entrapment:
 Fast method of immobilization
 Cheap (low cost matrixes available)
 Easy to practice at small scale
 Mild conditions are required
 Less chance of conformational changes in enzyme
 Can be used for sensing application
 Disadvantages of entrapment:
 Leakage of enzyme
 Pore diffusion limitation
 Chance of microbial contamination
 Not much success in industrial process

13. MicrocapsuleType Entrapmet/
Encapsulation/Membrane Confinement
 It involves enclosing the enzymes within semi -
permeable polymer membranes e.g. semi permeable
collodion or nylon membranes in the shape of spheres.

14. ADVANTAGES DISADVANTAGES
 1. No chemical
modification.
 2. Relatively stable
forms.
 3. Easy handling and re-
usage.
 1. The enzyme may leak
from the pores.

15. Chemical Method: Covalent
Binding
 Based on the binding of enzymes and water-insoluble carriers by covalent
bonds
 The functional groups that may take part in this binding are Amino
group, Carboxyl group, Sulfhydryl group, Hydroxyl group, Imidazole
group, Phenolic group, Thiol group, etc
 Disadvantages : covalent binding may alter the conformational structure
and active center of the enzyme, resulting in major loss of activity and/or
changes of the substrate
 Advantages : the binding force between enzyme and carrier is so strong
that no leakage of the enzymes occurs, even in the presence of substrate or
solution of high ionic strength.

16. Important functional groups of the enzyme that provide
chemical groups to form covalent bonds with support or carrier
 1. Alpha carboxyl group at ‘C’ terminal of enzyme
 2. Alpha amino group at ‘N’ terminal of enzyme
 3. Epsilon amino groups of Lysine and Arginine in the enzyme
 4. β and γ carboxyl groups of Aspartate and Glutamate
 5. Phenol ring of Tyrosine
 6. Thiol group of Cysteine
 7. Hydroxyl groups of Serine and Threonine
 8. Imidazole group of Histidine
 9. Indole ring of Tryptophan

17. Carriers or supports commonly used for covalent
bonding are:
 Carbohydrates: Cellulose, DEAE cellulose, Agarose
 Synthetic agents: Polyacrylamide
 Protein carriers: Collagen, Gelatin
 Amino group bearing carriers: Eg. amino benzyl
cellulose
 Inorganic carriers: Porous glass, silica
 Cyanogen bromide (CNBr)-agarose and CNBr
Sepharose

18. Methods of covalent bonding
 (1). Diazoation: Bonding between amino group of support and
tyrosil or histidyl group of enzyme.
(2). Peptide bond: Bonding between amino or carboxyl groups
of the support and that of the enzyme.
(3). Poly functional reagents: Use of a bi-functional or
multifunctional reagent (glutaraldehyde) which forms covalent
bonds between the amino group of the support and amino group
of the enzyme.

19. Advantages of covalent bonding: Disadvantages of covalent bonding
(major problem with covalent
bonding):
(a). Strong linkage of enzyme
to the support
(b). No leakage or desorption
problem
(c). Comparatively simple
method
(d). A variety of support with
different functional groups
available
(e). Wide applicability
(a). Chemical modification of enzyme
leading to the loss of functional
conformation of enzyme.
(b). Enzyme inactivation by changes in
the conformation when undergoes
reactions at the active site. This can be
overcome through immobilization in
the presence of enzyme’s substrate or
a competitive inhibitor.

20. 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.
 This method is also called as
copolymerization

21.  Commonly used polyfunctional reagents are glutaraldehyde and
diazonium salt.
 This technique is cheap and simple but not often used with pure
enzymes.
 This method is widely used in commercial preparations and
industrial applications.
 The greatest disadvantage or demerit of this method is that the
polyfunctional reagents used for cross linking the enzyme may
denature or structurally modify the enzyme leading to the loss of
catalytic properties.

22. 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.

23. Fig. Pictorial representation of different immobilization methods.

24. Immobilization of whole cells:
 Immobilization of whole cells is an alternative to enzyme immobilization and it is a
well-developed method for the utilization of enzymes from microbes.
 Immobilization of whole cells become particularly effective when the individual
enzymes become inactive during direct immobilization, or the isolation and
purification of enzyme is not cost effective.
 The greatest advantage of whole cell immobilization is that here the enzymes will
be active and stable for long period of time since they are in their natural
environment.
 Use of immobilized cells for fermentation is a very old practice. Bacteria or yeast
cells are immobilized by adsorbing it on woodchips. This is practiced in many parts
for different types of fermentations.

25. Advantages of whole cell
immobilization:
Disadvantages of whole cell
immobilization:
(a). Multiple enzymes can be
introduced to a single step
(b). Extraction and purification of
enzymes are not required
(c). Enzymes are stable for long time
(d). Native conformation of enzyme is
best maintained
(e). Cell organelles like mitochondria
and chloroplasts can be immobilized
(f). Cost effective method
(a). Concentration of enzymes will be
less
(b). Production of unwanted enzymes
and unwanted products
(c). Modification of end products by
other enzymes produced by
immobilized cells

26. Methods of immobilization of whole cells:
 Methods of immobilization of whole cells are same as that
described for enzyme immobilization and they include:
 Adsorption
 Covalent bonding
 Cell to cell cross linking
 Encapsulation
 Entrapment

27. Immobilization of cells: Methods, Support materials, Cells
and Reaction:

28. Comparison Between The Methods

29. Limitations Of Enzyme
Immobilization
 Cost of carriers and immobilization.
 Changes in properties (selectivity).
 Mass transfer limitations.
 Problems with cofactor and regeneration.
 Problems with multienzymes systems.
 Activity loss during immobilization.

30. Thank you….