“Enzyme immobilization may be defined as confining the enzyme molecules to a distinct phase from the

one where in the substrate and product are present.”

• An immobilized enzyme is an enzyme that is attached to an inert, insoluble material such as calcium
alginate
• This can provide increased resistance to changes in conditions such as pH, temperature and several
environmental factors.
• It also allows enzymes to be held in place throughout the reaction, following which they are easily
separated from the products and may be used again a far more efficient process and so is widely used in
industry for enzyme catalyzed reactions.
• An alternative to enzyme immobilization is whole cell immobilization.
Salient feature of enzyme immobilization
• Enzymes are more or less physically confined in the course of a definite continuous catalytic process.
They may be suitably recovered from the reaction mixture and used over and over again there by
gainfully improving the economic viability of the entire process.
• It may be accomplished by fixing the enzyme molecules to or within certain appropriate substance.
• It should be absolutely critical that both the substrate and the products migrate quite freely in and out
of the phase to which the specific molecules are actually confined.
• Certain enzymes which are thermolabile in nature could be made heatstable by attachment into inert
polymeric supports
• Immobilized enzymes may be recycled, rapidly controlled, operated continuously, product(s) easily
separable, above all the enzyme (i.e., stability, pH) are altered favourably.

Methods of immobilization
1. Carrier binding. 1. Physical adsorption. 2. Covalent bonding. 3. Ionic bonding.
2. Cross linking.

3. Entrapment. 1. Occlusion within a cross linked gel. 2. Microencapsulation

. Physical adsorption
• This method is based on the physical adsorption of enzyme on the surface of water-insoluble carriers.
Like ion-exchange matrices, porous carbon, clay, hydrous metal oxides, glasses and polymeric aromatic
resins.
• The bond between the enzyme and carrier molecule may be ionic, covalent, hydrogen, coordinated
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.
• Advantages of adsorption:- • Little or no confirmation change of the enzyme. • Simple and cheap. • No
reagents are required. • Wide applicability and capable of high enzyme loading.
• Disadvantages of adsorption:- • Desorption resulting from changes in temperature, pH, and ionic
strength. • Slow method.

Covalent bonding • Covalent binding is the most widely used method for immobilizing enzymes. The
covalent bond between enzyme and a support matrix forms 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 a cellulose-based support with cyanogen bromide, which is
then mixed with the enzyme.
• Advantages of covalent coupling:-
• Strength of binding is very strong, little or no leakage from the support
• This is a simple, mild and often successful method of wide applicability
• Disadvantages of covalent coupling:-
• Enzymes are chemically modified and can denatured during immobilization
• Small amounts of enzymes may be immobilized (about 0.02 g/g of matrix)

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
. • The most common reagent used for cross-linking is glutaraldehyde.
• Advantages of cross linking:-
• Very little desorption (enzyme strongly bound)
• Best used in conjunction with other methods.
• Disadvantages of cross linking:-
• May cause significant changes in the active site.

Entrapment
• In entrapment, the enzymes 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 a chemical reaction.
• Polymers like polyacrylamide, collagen, cellulose acetate, calcium alginate or carrageenan etc. are
used as the matrices.
• Advantages of entrapment:- • Loss of enzyme activity upon immobilization is minimized.
• Disadvantages of entrapment:- • The enzyme can leak into the surrounding medium. • Another
problem is the mass transfer resistance to substrates and products. • Substrate cannot diffuse deep into
the gel matrix
Advantages of 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
• Increased functional efficiency of the enzyme
• Minimum reaction time and cost effective
• Less labour input in the process
Disadvantages of immobilization
• It gives rise to an additional bearing on cost for isolation, purification and recovery of active enzyme.
• It invariably affects the stability and catalytic 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.
• Industrial applications are limited as very few companies are using immobilized enzymes or whole
cells.
• Enzymes are inactivated by the heat generated in the system.
• Some enzymes became unstable after immobilization.

Applications
1. Industrial production: Industrial production of antibiotics, beverages, amino acids etc. uses
immobilized enzymes.
2. Biomedical applications: Immobilized enzymes are widely used for diagnosis and treatment of many
diseases such as inborn disorder.
3. Food industry: Enzymes like pectinases and cellulases immobilized on suitable carriers are
successfully used in the production of jams, jellys and syrups from fruits and vegetables.
4. Research: The use of immobilized enzyme allow researcher to increase the efficiency of different
enzymes such as different proteases for cell and organelle lysis.
5. Biodiesel production from vegetable oils.
6. Textile industry: Scouring, bio polishing and desizing of fabrics.
7. Waste water management: Treatment of sewage and industrial effluents. 8. Detergent industry:
Immobilization of lipase enzyme for effective dirt removal from cloths.