Lect.

Immobilization
techniques
Introduction
 What Is Enzyme Immobilization?
• Enzyme immobilization may be defined as : a
process of confining the enzyme molecules to
solid support over which a substrate is passed
and converted to products.
• The process whereby the movement of enzymes,
cells, organelles, etc. in space is completely or
severely restricted usually resulting in water
insoluble form of the enzyme
• The Immobilized Enzyme: is one whose
movement in space has been restricted either
completely or to a small limited region.
 Need for Immobilization
• Protection from degradation and deactivation.
• Retention of enzyme, enzyme – free products.
• Recycling, repetitive use
• Cost efficiency.
• Enhanced stability.
• Use as controlled release agents.
• The ability to stop the reaction rapidly by
removing the enzyme from reaction solution (or
vice-verse)
• Allows development of multi-enzyme reaction
system.
 Advantages of Immobilized enzyme

• Stability of the enzyme increase.

• Can be used again and again
• Products are enzyme free.
• Can be used for continuous product
formation.
• Suitable for industrial and medical use.
• Minimize effluent disposal problems.
Disadvantages of Immobilized enzyme
• It gives rise to an additional bearing on cost.
• It invariably 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.
• Certain immobilization protocols offer serious
problems with respect to the diffusion of the
substrate to have an access to the enzyme.
 Carrier Binding –Physical Adsorption
• Binding of the enzyme to the surface of an
insoluble carrier that has not been specifically
functionalized for covalent coupling.
• Examples of suitable adsorbents are ione
exchange matrices, porous carbon, clay, hydrous
metal oxides, glasses and polymeric aromatic
resins.
• The bond between the enzyme and carrier
molecules may be ionic, covalent, hydrogen,
coordinate covalent or even combination of any
of these.
• Immobilization can be brought about by
coupling an enzyme either to external or
internal surface of the carrier.
• The external surface binding method is
advantageous as it does not involve conditions
like pore diffusion.
• The disadvantages however, include exposure of
enzymes to microbial attack physical abrasion of
enzyme due to turbulence associated with the
bulk solution.
• The major disadvantage of the internal
immobilization method is the pore diffusion.
 Factors affecting adsorption
1. pH
• The pH will be a very major parameter which will affect the
binding in the case of ionic binding.
• Even for other forms of binding processes pH plays a key
role and very often it has been noted that the binding by
adsorption is maximum at its isoelectric points.
2. Salts
At high salt concentration adsorption is usually poor and
minimum salt concentration is required to be kept.
3. Protein concentration
The definite ratio of protein to carrier will play a particular
equilibrium behavior and higher the protein concentration
you can reach nearer the saturation level at equilibrium
Temperature.
4. Temperature
• increasing the temperature reduces the adsorption,
• when we are talking about enzymes adsorption over a
narrow range of temperature offers a reverse phenomena.
• That means adsorption increases with temperature, “over a
narrow range of temperature increase”. If you increase to a
higher level desorption also might start.
• we consider the adsorption at …… range of temperature.
The primary factors responsible for the increase in
adsorption with temperature are when the protein binds to
a surface there is some unfolding of the protein molecules,
which is an endothermic process and the increase of
temperature resulting in enhanced adsorption
• The other factor which is important, which is probably
attributing to this increase of adsorption with the increase in
temperature is the diffusional processes
4. Carrier properties,
depending on the property like surface property of
the carrier particles which were used as matrix,
will also play a role on the adsorption.
5. Time of adsorption
The diffusion is involved and the time required
very often is controlled by diffusional limitations
and the rate of diffusion controls the time required
for adsorption process.
Methods of immobilization by adsorption
• The absorptive immobilization of enzymes can be
done by following methods:
1. Static process: this is most efficient technique but
requires maximum time. 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
admixing enzyme with carrier under constant
agitation using mechanical shaker.
3. Reactor Loading: this process is employed for the
commercial production of the immobilized
enzymes. The carrier is placed into the reactor and
enzyme solution is transferred to the reactor with
agitation of the whole contents in the reactor.
4. Electro –Deposition
In this technique, carrier is placed in the vicinity of
one of the electrode in an enzyme bath and
electric current applied leading to migration of
enzyme towards the carrier. This results in
deposition of enzyme on the surface of the carrier.
 Covalent Bonding
• Covalent binding is the most widely used
method for immobilizing enzymes.
• The covalent bond between enzyme and a
support matrix form a stable complex.
• The functional group present on enzyme,
through which a covalent bond with support
could be established, should be non essential for
enzymatic activity.
• The most common technique is to activate
cellulose –based support with cyanogen
bromide, which is then mixed with the enzyme.
• The protein functional group which can be
utilized in covalent coupling include:
• Amino group.
• Carboxylic group.
• Phenol ring.
• Indole group.
 IONIC BONDING
• The enzyme is made to bind to the support through
ionic bonding
• DEAE sephadex, DEAE cellulose, Dollen -
50,carboxymethyl cellulose and amberlite are the
supports usually used.
 CROSS LINKING
• This method is based on the
formation of covalent bonds
between the enzyme molecules
by means of multifunctional
reagents leading to three
dimensional cross linked
aggregates.
• It is used mostly as a means of
stabilizing adsorbed enzymes
and also for preventing leakage
from polyacrylamide gels.
 Entrapment
• In entrapment, the enzyme or cells are not directly
attached to the support surface, but simply trapped
inside the polymer matrix.
• Entrapment is carried out by mixing the biocatalyst
into a monomer solution, followed by polymerization
initiated by a change in temperature or by chemical
reaction.
• Polymers like Polyacrylamide, collagen, cellulose
acetate, calcium alginate or carrageenan etc. are used
as the matrices
Entrapment Classification
 Types of Entrapments
• 1. Lattice type entrapment
• 2. Encapsulation
Thank you