Enzyme and cell immobilization involves trapping enzymes or cells in a support matrix, enhancing stability and reusability while permitting substrate exchange. Techniques for immobilization include carrier binding, cross-linking, and entrapment, each with specific advantages and disadvantages. Despite benefits like multiple uses and easier separation from products, challenges such as cost, potential activity loss, and limited industrial applicability exist.

1. ENZYME AND CELL
IMMOBILIZATION

2. Definition of Enzyme/Cell
Immobilization
The process where imprisonment of enzyme or cell is done
in a distinct support or matrix due to which movement of
enzymes in space is completely or severely restricted.
Support or matrix allows exchange of medium containing
substrate.
FACTS
• Immobilized enzymes/ cells are also sometimes referred
to as insolubilized, supported or matrix-linked
enzymes/cells.

3. Need of Enzyme Immobilization-
1.Economic Nature-
In most of the processes, enzymes are mixed in a solution with
substrates and cannot be economically recovered after the
reaction and are generally wasted. This problem can be overcome
by using enzymes in an immobilized or insolubilized form so that
they can be retained in a biochemical reactor for further catalysis.
2. Increased Stability-
Immobilized enzymes typically have
greater thermal and operational stability than the soluble form of
the enzyme. Enzymes are fixed to an inert carrier in most stable
functional form and sites for sensing the variation in temperature
and pH are blocked by immobilization.

4. Need for immobilizing cells
• Immobilized enzymes are not suitable for
multienzyme reactions. The whole cells or
cellular organelles can be immobilized to serve
as multi-enzyme systems.
• Extracted intracellular enzymes are unstable.
So, whole cell is immobilized.

5. Materials used for fabrication of
immobilization supports
• Alginate, Chitin, Collagen,
Gelatin, Cellulose, Starch,
Pectin
Natural
Polymers
• DEAE cellulose, Polyvinyl Chloride,
Polyaniline, glutaraldehyde
activated nylon, UV- activated
polyethylene glycol
Synthetic
Polymers
• Zeolites, Ceramics, Celite,
silica, Activated carbon, Glass,
Charcoal
Inorganic
Materials

6. TECHNIQUE OF ENZYME/CELL
IMMOBILIZATION:-
The techniques employed for immobilization of cells are almost the
same as that used for immobilization of enzymes
1. Carrier binding.
• Physical adsorption.
• Covalent bonding.
• Ionic bonding.
2. Cross linking
3. Entrapment
• Inclusion in- gel ; fiber ; microcapsules
• Microencapsulation.

7. 1. Carrier Binding: a) Physical Adsorption
• This method is based on the physical adsorption of enzyme
protein on the surface of water-insoluble carriers like silica gel,
calcium phosphate gel, glass, starch, DEAE cellulose.
• The bond between the enzyme and carrier molecule may be
ionic, covalent, hydrogen, coordinated covalent or even
combination of any of these.

8. • Coupling of enzyme can be done either to external or
internal surface of the carrier.
i. External surface binding method is advantageous as it
does not involve conditions like pore diffusion. The
disadvantages include exposure of enzymes to
microbial attack, physical abrasion of enzyme due to
turbulence associated with solution.
ii. Internal Surface major disadvantage is the pore
diffusion.
• Adsorbed enzymes can be easily removed by minor
changes in pH, ionic strength or temperature. This is a
disadvantage for industrial use of enzymes.

9. Methods of immobilization by
physical adsorption-
1. Static Process:- In this technique, enzyme is immobilized by
allowing it to be in contact with the carrier without agitation.
2. Dynamic Process:- This process typically involves the mixing of
enzyme with the carrier under constant agitation using mechanical
shaker.
3. Reactor loading:- This process is employed for commercial
production of immobilized enzymes. Carrier is placed in reactor and
enzyme solution is transferred to reactor with agitation of the
whole content in the reactor.
4. Electro-Deposition:-Carrier is placed in vicinity of one of the
electrode in an enzyme bath and electric current is applied leading
to migration of enzyme towards the carrier. This results in
deposition of enzyme on the surface of the carrier

10. 1. Carrier Binding: b) Covalent Bonding
• Immobilization of the enzymes
can be achieved by creation of
covalent bonds between the
chemical groups of enzymes
and the chemical groups of the
support. However, covalent
binding is often associated with
loss of some enzyme activity.
The inert support usually
requires pretreatment (to form
pre-activated support) before it
binds to enzyme.

11. Methods of Covalent Binding
1. Cyanogen bromide activation: The inert support materials
(cellulose, sepharose, sephadex) containing glycol groups are
activated by CNBr (Cyanogen Bromide) which bind to enzymes.
2. Diazotation: Some support materials (amino benzyl cellulose,
amino derivatives of polystyrene) are subjected to diazotation on
treatment with NaNO2 and HCI. They, in turn, bind covalently to
tyrosyl or histidyl groups of enzymes.
3. Peptide bond formation: Formation of peptide bonds between
the amino (or carboxyl) groups of the support and the carboxyl (or
amino) groups of enzymes.
4. Activation by bi- or poly-functional reagents: Some of the
reagents such as glutaraldehyde can be used to create bonds
between amino groups of enzymes and amino groups of support (
aminoethylcellulose, albumin, amino alkylated porous glass).

13. 2.CROSS LINKING
• The absence of a solid support is a
characteristic feature of
immobilization of enzymes by
cross- linking. The enzyme
molecules are immobilized by
creating cross-links between them,
through the involvement of poly-
functional reagents. Reagents
react with the enzyme molecules
and create bridges which form the
backbone to hold enzyme
molecules.
• Reagents include-glutaraldehyde,
diazobenzidine, hexamethylene
diisocyanate.

14. • Glutaraldehyde is the most extensively used cross-linking
reagent. It reacts with lysyl residues of the enzymes and forms a
Schiff’s base. The cross links formed between the enzyme and
glutaraldehyde are irreversible and can withstand extreme pH
and temperature.
Eg. glucose isomerase, penicillin amidase are immmobilized by
glutaraldehyde
• Advantage- The technique of cross-linking is quite simple and
cost-effective.
• Disadvantage- It involves the risk of denaturation of the enzyme
by the poly-functional reagent.

15. 3. Entrapment
• In entrapment, the enzymes or cells are not directly attached to the support
surface, but simply trapped inside the polymer matrix. The size of the matrix
pores is such that the enzyme is retained while the substrate and product
molecules pass through but disadvantage is that low molecular weight
enzymes are lost In this technique the enzyme is not subjected to strong
binding forces and structural distortions but some deactivation may occur due
to changes in pH or temperature or addition of solvents.
• The matrices used- polyacrylamide gel, collagen, gelatin, starch, cellulose,
silicone and rubber. Enzymes can be entrapped by several ways.

16. a. Methods of Entrapment
• Inclusion in Gels- Enzymes
trapped inside the gels.
• Inclusion in Fibers-
Enzymes supported on
fibers made of matrix
material.
• Inclusion of
Microcapsules- Enzymes
are trapped inside a
microcapsule matrix
formed by monomer
mixture such as polyamine
and calcium alginate.

17. b. Microencapsulation-
• It is type of entrapment . It is done by enclosing the enzymes in a
spherical membrane capsule. Capsule will be made of semi
permeable membrane that is impermeable to enzymes and
other molecules but is permeable to substrate and product. like
nitro cellulose or nylon. Effectiveness depends on stability of
enzymes inside capsule.

18. Advantages of Enzyme Immobilization
• Multiple or repetitive use of a single batch of enzymes.
• Immobilized enzymes are usually more stable.
• Ability to stop the reaction rapidly by removing the
enzyme from the reaction solution.
• Product is not contaminated with the enzyme.
• Easy separation of the enzyme from the product.
• Allows development of a multienzyme reaction system.
• Reduces effluent disposal problems

19. Disadvantages of Enzyme
Immobilization
• It gives rise to an additional bearing on cost for
purification and recovery of enzyme.
• It affects the stability and activity of enzymes.
• The technique may not prove to be of any
advantage when one of the substrate is found to
be insoluble.
• Catalytic properties of some enzymes are
reduced or completely lost after immobilization.
• Industrial applications are limited.
• Enzymes are inactivated by heat generated in
system.

20. Example of Immobilized Cells with
methods-