This document discusses enzyme immobilization, which refers to anchoring enzymes to an inert support to make them more stable and reusable. There are several advantages to immobilized enzymes such as increased efficiency and stability. Common immobilization methods include adsorption, entrapment, covalent binding, and cross-linking. Adsorption uses weak bonds to attach enzymes while covalent binding forms stronger covalent bonds. Immobilized enzymes have many applications including producing commercial products like amino acids and high fructose syrup as well as uses in analytical techniques and industrial processes.

1. Enzyme Immobilization
By S.D.Mankar
Assistant Professor
Department Of Pharmaceutics
Pravara Rural College Of Pharmacy,Pravaranagar
S.D.Mankar
1

2. Enzyme in free solutions ( i.e. in soluble or free form) react with
substrates to result in products. Such use of enzyme is wasteful,
since enzymes are not stable, and they cannot be recovered for
reuse.
Immobilization of enzyme refers to the technique of
anchoring the enzymes in or on an inert support for their
stability and functional reuse.
By employing this techniques enzymes are made more efficient
and cost effective for their industrial use.
Immobilized enzymes retain their structural conformation
necessary for catalysis.

3. Advantages:-
Stable and more efficient in function.
Can be reused again & again.
Products are enzyme free.
Ideal for multi-enzyme reaction systems.
Control of enzyme function is easy.
Suitable for industrial & medical use.
Minimize effluent disposable problems.

4. Disadvantages:-
Possibility of loss of biological activity of an enzyme during
immobilization or while it is in use.
Expensive
Required sophisticated equipments.

5. Immobilized enzymes are generally preferred over immobilized
cells due to specificity to yield the products in pure form.
Methods of Immobilization:-
Adsorption
Entrapment- microencapsulation.
Covalent binding
Cross linking

6. Adsorption:-
 adsorption involves the physical binding of enzyme on
the surface of an inert support.
The support materials may be inorganic ( e.g. alumina, silica
calcium phosphate gel, glass) or organic ( starch, CMC, DEAE-
cellulose, DEAE-sephadex)
Adsorption of enzyme molecules ( on the inert support)
weak forces such van der waals forces and hydrogen bonds.
Therefore, adsorbed enzymes can be easily removed by minor
changes in pH, ionic strength or temp.
This is disadvantage for industrial use of enzymes.

7. Entrapment:- enzymes can be immobiliozed by physical
entrapment inside a polymer or a gel matrix.
The size of the matrix pores is such that the enzyme is retained
while the substrate and product molecules pass through.
In this technique, commonly referred to as lattice entrapment.
Some deactivation may however, occur during immobilization
process due to changes in pH or temp. or addition of solvents.
The matrices used for entrapping of enzymes include
polyacrylamide gel, collagen, gelatin, starch, cellulose,
rubber.

8. Enzymes can be entrapped by several ways
1. Enzyme inclusion in gels:-entrapment of enzyme inside the
gels.
2. Enzyme inclusion in fibres:- enzymes are trapped in a fibre
format of the matrix.
3. Enzyme inclusion in microcapsules:- the hydrophobic &
hydrophilic forms of the matrix polymerise to form a
microcapsules containing enzyme molecules inside.
Major limitation- leakage of enzyme from matrix.

9. Microencapsulation:-
Is type of entrapment.
It refers to the process of spherical particle formation wherein
liquid or suspension is enclosed in a semipermeable
The membrane may be polymeric, lipoidal, lipoprotein based
non-ionic in nature.
There are 3 distinct ways
1. building of special membrane reactors.
2.Formation of emulsion.
3.Stabilization of emulsions to form microcapsules.

10. Pancreatic cells grown in cultures can be immobilized by
microencapsulation.
E.g hybridoma cells.
Covalent binding:-
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.
This techniques is widely used.
But chance of loss of enzyme activity.
Inert support required pre-treatment before it binds to enzyme.

11. 1. cyanogen bromide activation:-
The inert support materials(cellulose, sepharose,sephadex)
containing glycol groups are activated by CNBr, which then
to enzymes & immobilize them.
2.Diazotation:-
Some of the support materials are subjected to diazotation on
treatment with NaNO2 HCL.
They in turn bind covalently to tyrosyl or histidyl group of
enzymes.

12. 3. Peptide bond formation:-
Formation of peptide bonds between amino or carboxyl group
of the support & carboxyl or amino group of enzymes.
4. Activation by bi- or polyfunctional reagents:-
Some of the reagents such as glutaraldehyde can be used to
create bonds between amino groups of enzymes and amino
groups of support.

13. Cross-linking:-
The absence of solid support is a characteristic feature of
immobilization of enzymes by cross linking.
The enzyme molecule are immobilized by creating cross-links
between them, through the involvement of polyfunctional
reagents.
These reagents in fact react with the enzyme molecules and
create bridges which form the backbone to hold enzyme
molecules.
Glutarldehyde, diazobenzidine, hexamethylene di-isocyanate
tolune di-isothicyanate generally used.

14. Glutaraldehyde is most commonly used.
It reacts lysyl residues of the enzymes and forms Schiff’s base.
The cross link formed are irreversible and can withstand extreme
pH & temp.
Simple & Cost effective technique
But risk of denaturation of the enzyme by ployfunctional
reagent.
Choice of immobilization techniques based on the trial & error
approach to choose the ideal one.

15. Applications:-
1. Manufacture of commercial products:-
Production of L-amino acids:- aminoacylase can selectively
hydrolyse D,L- acyl amino acid to produce L-amino acids.
Production of high fructose syrup- produced from glucose by
employing an immobilized enzyme glucose isomerase.
2.Analytical applications:-
In biochemical analysis:- used for development of precise and
specific analytical techniques for the estimation of several
biochemical compounds.

16. In affinity chromatography and purification:-
Purify several compunds e.g antigens, antibodies, cofactors.
3. Industrial application:-
Used in production of pharmaceutical sub. Such as antibiotics,
steroids, & amino acids.

17. References:-
 1. Biotechnology by U.Satayanaryana (reference book)
pg no:-288-297.
2.Pharmacutical biotechnology by Dr.Chandrakant Kokare,
prakashan, pg no:-12.14-12.17.