Immobilisation.pdf

A brief Outlook on Immobilisation,methods,Advantages & Disadvantages

1. Immobilisation
Dr Sumitha J
Associate Professor
JBAS College for Women,Chennai-18

2. Immobilisation is defined as the
imprisonment of a cell or enzyme in distinct
support or matrix which allows the exchange
of medium containing substrate or effector
or inhibitor molecules

3. Advantages of immobilised enzymes
Increased functional efficiency of enzyme
Enhanced reproducibility of the process they are
undertaking
Reuse of enzyme
Continuous use of enzyme
Less labour input in the processes
Saving in capital cost and investment of the process

4. Advantages of immobilised enzymes
Minimum reaction time
Less chance of contamination in products
More stability of products
Stable supply of products in the market
Improved process control
High enzyme-substrate ratio

5. Methods of Immobilisation
Based on bond types and matrix used, Immobilisation may be grouped into 5 types.
Physical Methods Chemical Methods
Adsorption
Entrapment
Encapsulation
Cross Linking
Covalent Bonding

6. Adsorption
In this type, the enzyme adheres to the surface of the water-insoluble
carrier matrix.
The binding is nonspecific like electrostatic or hydrophobic affinity
binding to a particular ligand.
The binding between enzymes and the carrier matrix is usually firm, but
it gets weakened by factors like the Addition of substrate and pH or
ionic strength
In enzyme adsorption, the bonding is non-permanent and accomplished
by weak bonds, mainly hydrogen bonds and Vander Waal forces.
The matrix-500A-1mm
Different Carrier Materials used-Aluminium Oxide, Clay and Starch
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7. Methods of Immobilization by Adsorption
Static method: It involves the immobilization of enzyme and carrier
molecules without agitation.
Dynamic process: It involves the mixing of an enzyme with the carrier,
under constant agitation.
Reactor loading: It involves transferring both enzyme and carrier in the
reactor with the agitation of the whole content. It is widely used for the
commercial production of immobilised enzymes.
Electro-deposition: Here, a carrier is kept proximal to the electrode in an
enzyme bath, after which an electric current is passed through it. It results
in the movement of an enzyme towards the carrier. At last, the enzyme gets
deposited on the surface.

8. Advantages of Adsorption
no pore diffusion limitation.
simple and economical method
limited loss of enzyme activity.
less disruption to an enzyme.
minimum activation steps.
The adsorbed enzyme can be
recycled, regenerated and reused.
high loading efficiency of an
enzyme.
Dis-Advantages of Adsorption
low surface area for enzyme binding.
Desorption of an enzyme from the
carrier usually occurs.
the yield is low

9. Methods of Immobilization by
Entrapment
It involves the inclusion of an enzyme
into the following matrices:
Gels: Involves entrapment of an enzyme
inside the gel matrix.
Fibres: Entrapment of an enzyme inside
the fibre matrix.
Microcapsules: Involves entrapment
inside a microcapsule.

10. Entrapment
An enzyme traps inside a porous
polymer or gel matrix during this
method.
It is also called lattice entrapment. 
The bonding between an enzyme and
matrix can be covalent or non-covalent.
Matrix used-PAGE, Cellulose acetate,
agar, gelatin, alginate
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11. Advantages of Entrapment
enzyme loading capacity is
high.
rapid & easy method.
the enzyme distortion is low.
Dis-Advantages of Entrapment
Low rate of diffusion of substrate and
product .
leakage of low molecular weight
enzymes.
chances of microbial contamination.
causes enzyme inactivation and
sometimes loss of enzyme activity.
limited industrial use.

12. Encapsulation
It is the membrane confinement method.
An enzyme confines within the semipermeable
membrane of a capsule in an aqueous solution.
This process allows the exchange of medium (substrate
& product), but not the enzyme.
The effectiveness of encapsulation relies upon the
enzyme stability.
The capsule is made of a semi-permeable membrane,
and it can be polymeric, lipoid, non-ionic etc. in nature.
Different Carrier Materials used-nitrocellulose, nylon

13. Methods of Immobilisation by Encapsulation
Encapsulation in a reaction vessel: It involves the partitioning of a
chamber by a semipermeable membrane. One chamber contains
enzymes, whereas the other contains substrate and product.
Encapsulation by hollow fibre membrane: It involves entrapment of
an enzyme inside a semipermeable matrix (cellulose, triacetate etc.).
Here, an enzyme traps inside the space of the matrix.
Microencapsulation: By chemical polymerization, the enzyme
molecules enclose inside a microcapsule by the use of 1-6-
diaminohexane.
Encapsulation by liposomes: Here, an enzyme binds to the concentric
lipoidal membrane of the liposome by the use of phospholipid.

14. Advantages of Encapsulation
no enzyme leakage.
simple method to conduct.
high loading efficiency of an
enzyme.
Dis-Advantages of Encapsulation
the use of a carrier that has a
pore size limitation.
not so cost-effective

15. Covalent Binding
An enzyme molecule binds to the carrier by a covalent bond
The binding strength is powerful
The bonding is stable.
no enzyme loss during the process.
Covalent binding occurs between the active part, i.e. the functional group of
an enzyme and the carrier molecule.
The functional group that are participating in the binding process are
-NH2, -NH3, -COO, -OH, -SH, -O, -S etc.
The order of reactivity of these functional groups to the carrier depends
upon their charged status:
-S– > -SH > -O – > -NH2 > -COO – > -OH >> -NH
Carrier Molecules celluloses, agarose, sepharose, polyvinyl alcohol, silica and
porous glasses.


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16. Methods of Immobilisation by Covalent Bonding
Diazotation: bonding between the amino group of the matrix
and tyrosyl or histidyl group of an enzyme on the reaction
with NaNO2 and HCl
By peptide bond: bonding between amino or carboxyl group
of the matrix to the carboxyl or amino group of an enzyme.

Cyanogen bromide activation: binding of glycol groups of a
matrix with an enzyme by the activation of CNBr.

By polyfunctional reagents: bonding between the amino
group of the matrix and amino group of an enzyme. Example:
Glutaraldehyde (Bi-functional reagent).

17. Advantages of Covalent Binding:
high binding strength between an
enzyme and a carrier.
no enzyme leakage.
simple and widely used method.
not affected by the pH or ionic
strength.
Dis-Advantages of Covalent Bonding
Denaturation of the enzyme occurs
during the immobilization.
Only a small amount of enzyme can
be immobilized.
It is not so cost-effective.

18. Cross linking/Copolymerization
The immobilized enzymes covalently link to the
various groups of an enzyme via polyfunctional
reagents.
It does not require a support matrix.
Cross-linking leads to the formation of 3D
crosslinked aggregates.
The most commonly used polyfunctional agents are
glutaraldehyde and diazonium salts etc.

19. Advantages of Cross Linking
no enzyme leakage.
highly stabilized.
simple and cheap method.
wide applicability in the
commercial production of the
enzyme
Dis-Advantages of Cross linking
It causes enzyme inactivation.
cause enzyme denaturation.
It is not so cost-effective

20. Application
of
Immobilised
Enzymes
Research
Biodiesel
Textile
Waste Water Management
Textile
Detergent
Textile
Food
Biomedical Application