1. P R E S E N T E R : M A N Z A R A L I
R O L L N O : F 1 6 C H 1 1 5
D ES C R I B E E N Z Y ME I M M O B I L I Z AT I O N T E C H N I QU ES D E M O N ST R AT I N G O N E
E X A MP L E O F E N Z Y ME I M M O B I L I Z AT I O N I N E A C H T E C H N I QU E
S U B J E C T : B I O C H E M I C A L E N G I N E E R I N G
F - 1 6 B A T C H , C H E M I C A L E N G I N E E R I N G
T E A C H E R : P R O F . D R . A Z I Z A A F T A B M E M O N
D A T E D : J U L Y 23, T H U R S D A Y 2 0 2 0
.
2. Introduction
Definition: Immobilization is defined as imprisonment of
enzyme in a support of matrix
It is a process of confining the enzyme molecule to a solid
support over which a substrate molecule is passed and
converted to a product.
Advantages of Immobilization Enzymes:
Protection from degradation and deactivation
Enhance stability of the product
Reuse of enzyme is possible, lowering the cost of
production of enzyme.
Chance of product contamination is less.
3. Enzyme Immobilization
Advantages of Immobilize Enzymes
1. Protection from degradation and deactivation.
2. Re-use of enzymes for many reaction cycles, lowering the total
production cost of enzyme mediated reactions.
3. Ability to stop the reaction rapidly by removing the enzyme
from the reaction solution.
4. Enhanced stability.
5. Easy separation of the enzyme from the product.
6. Product is not contaminated with the enzyme.
An Ideal Carrier Matrices For Enzyme Immobilization
Physically strong and stable.
Cost effective.
Regenerable, Reduction in product inhibition.
4. METHODS FOR ENZYME IMMOBILIZATION
There are several methods for enzyme immobilization such like
5. METHODS FOR ENZYME IMMOBILIZATION
Physical Methods For Immobilization:
ADSORPTION
o Involves the physical binding of the enzyme on the surface of carrier
matrix.
o Carrier may be organic or inorganic.
o The process of adsorption involves the weak interactions like Vander
Waal or hydrogen bonds.
o Carriers: - silica, bentonite, cellulose, etc.
o For example: catalase & invertase
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.
6. METHODS FOR ENZYME IMMOBILIZATION
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. 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.
Lattice-Type Entrapment
o Entrapment involves entrapping enzymes within the interstitial spaces of a
cross-linked water-insoluble polymer. Some synthetic polymers such as
polyarylamide, polyvinyalcohol, etc... and natural polymer (starch) have
been used to immobilize enzymes using this technique.
7. METHODS FOR ENZYME IMMOBILIZATION
Microcapsule Type
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.
ADVANTAGES
1. No chemical modification.
2. Relatively stable forms.
3. Easy handling and re-usage
DISADVANTAGES
1. The enzyme may leak from the
pores.
8. METHODS FOR ENZYME IMMOBILIZATION
Covalent Binding
It is a widely used method. In this system, enzyme molecule binds to the carrier by
a covalent bond. The binding strength is powerful. A complex form through this
bonding is stable. There is no enzyme loss during the process.
Covalent binding occurs between the active part, the, i.e. functional group of an
enzyme and carrier molecule. The functional group takes part in binding are -NH2,
-NH3, -COO, -OH, -SH, -O, -S etc. The order of reactivity of these functional group
to the carrier depends upon their charged status:
-S– > -SH > -O– > -NH2 > -COO– > -OH >> -NH3
+
Examples of a polymeric carrier used in this process are:
Carboxylic acid and related groups of polyglutamic acid
Amide group of a polypeptide
Amino and related groups of polysaccharides
Some most commonly used polymers are the polysaccharide (celluloses, agarose,
sepharose, etc.), polyvinyl alcohol, silica and porous glasses.
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.
9. METHODS FOR ENZYME IMMOBILIZATION
Cross Linking
1. Cross linking involves intermolecular cross linking of enzyme molecules in the
presence/absence of solid support.
2. It is also called “Copolymerization“. In this, immobilized enzymes covalently
link to the various groups of an enzyme via poly functional reagents. It does not
require a support matrix. Cross-linking leads to the formation of 3D crosslinked
aggregates.
Commonly used polyfunctional agents are Glutaraldehyde, diazonium salts etc.
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.
10. METHODS FOR ENZYME IMMOBILIZATION
Disadvantages of Immobilization
Industrial use is limited
Enzymes can lose their catalytic
property
Some proteins lose their stability
Enzymes inactivate by heat generation
Expensive to carry out
Advantages of Immobilization
Can be reused
Decrease the labour input
Can be used continuously
Increase enzyme and substrate ratio
Requires minimum activation time
11. METHODS FOR ENZYME IMMOBILIZATION
Applications of Immobilized enzymes
o Industrial use: E.g. Production of antibiotics, amino acids etc.
o
Biomedical use: E.g. For the treatment, diagnosis and drug delivery.
o In the food industry: E.g. Production of jams, jellies, syrups etc.
o Sewage treatment: Used in the treatment of sewage and industrial effluent for
wastewater management.
o In the detergent industry: E.g. Immobilization of lipases to digest lipid present
in stains or dirt.
o
Textile industry: E.g. Scouring, bio-polishing etc.
Thus immobilization process is widely used in all the fields where the enzyme is
immobilized to the particular phase where an enzyme can be reused and stabilize to
carry out many reactions.
