A simple description of immobilized enzymes and their perspectives.

1. Immobilized Enzyme
Md. Nafizur Rahman
Dept. of Genetic Engineering & Biotechnology,
Shahjalal University of Science & Technology, Sylhet.

2. What are enzymes and what do they do?
• Enzymes are proteins with highly specialized catalytic functions,
produced by all living organisms. OR “Enzymes are proteins, which
catalyze specific biochemical reactions in a very efficient manner.”
• Enzymes are biodegradable and responsible for many biochemical
reactions in micro-organisms, plants, animals, and human beings.
• Although like all other proteins, enzymes are composed of amino acids,
they differ in function in that they have the unique ability to facilitate
biochemical reactions without undergoing change themselves. This
catalytic capability is what makes enzymes unique.
• Enzymes are highly efficient in increasing the reaction rate of
biochemical processes that otherwise proceed very slowly, or in some
cases, not at all.

3. Enzyme immobilization
• Enzymes are most extensively used in the food and beverage industries
across the world to meet the increasing demand for nutritionally superb
and high-value products.
• However, the predominant use of the enzymes has been limited by the
fact that large number of these enzymes are unstable and the cost of
isolation, purification and recovery of the active enzyme is high.
• In actual practice, the soluble enzymes engaged in batch operations is
found to be uneconomical as the active enzyme is virtually lost (not
recovered) after each viable reaction.
• Therefore, in order to overcome such non productive, economically not
feasible, and deleterious effects the enzymes have been ultimately
immobilized and this process is termed as enzyme immobilization.

4. “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 (produced by reacting a
mixture of sodium alginate solution and enzyme solution with calcium
chloride).
• 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.

5. 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 heat-
stable 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.

6. 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.

7. Physical adsorption
• This method is based on the physical adsorption of enzyme on the
surface of water-insoluble carriers. Examples of suitable adsorbents are
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.

8. 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)

9. 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.

10. 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.

11. Table: Comparison between main enzyme immobilization methods

12. 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

13. • 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.
Disadvantages of immobilization

14. 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.
Applications of immobilization

15. Table: major products obtained using immobilized enzyme
Uses of Immobilized Enzymes
• Biotransformation
• Secondary metabolite production
• Biosensors
• Enzyme-linked immunosorbent assays (ELISAs)
• Biological washing Powders
• Food Industry
• Seed Germination

16. Reference
• http://www.easybiologyclass.com/enzyme-cell-immobilization-
techniques/
• http://enzymeimmobilization.blogspot.com/2011/02/enzyme-
immobilization.html
• http://www.scribd.com/doc/31429014/Immobilized-Enzymes
• Immobilization of Enzymes: A Literature Survey: Beatriz Brena , Paula
González-Pombo , and Francisco Batista-Viera

17. Thanks Everyone