immobilization of enzymes

2. Definition
 Enzyme immobilization may be defined as a process of
confining the enzyme molecules to a solid support
over which a substrate is passed and converted to
products.
 What is an immobilized Enzyme?
 An immobilized enzyme is one whose movement in
space has been restricted either completely or to a
small limited region.

3. Introduction
 Many enzymes secreted by microorganisms are available on a
large scale and there is no effect on their cost if they are used
only once in a process.
 In addition, many more enzymes are such that they affect the
cost and could not be economical if not reused.
 Therefore, reuse of enzymes led to the development of
immobilization techniques.
 It involves the conversion of water soluble enzyme protein
into a solid form of catalyst by several methods.
 It is only possible to immobilize microbial cells by similar
techniques

4. Introduction
 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 or temperature.
 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 catalysed
reactions.
 An alternative to enzyme immobilization is whole cell
immobilization.

5. Why immobilized enzymes?
 Protection from degradation and deactivation.
 Re-use of enzymes for many reaction cycles, lowering the total
production cost of enzyme mediated reactions.
 Ability to stop the reaction rapidly by removing the enzyme from
the reaction solution.
 Enhanced stability
 Improved process control
 Easy separation of enzymes from the product.
 Product is not contaminated with enzyme or less chance of
contamination in products

6. An Ideal carrier Matrices for enzyme Immobilization
 Inert.
 Physically strong and stable.
 Cost effective.
 Regenerable.
 Reduction in product inhibition.

7. Methods of Enzyme Immobilization
 There are five different techniques of immobilizing
enzymes placed in two types or groups.
 Physical
 (1) Adsorption
 (2) Entrapment
 (3) Encapsulation
 Chemical
 (4) Covalent binding
 (5) Cross- linking or copolymerization

9. Physical Adsorption
 Physical adsorption of an enzyme onto a solid is probably the simplest
way of preparing immobilized enzymes.
 An enzyme may be immobilized by bonding to either the external or
internal surface of a carrier or support such as mineral support ( alumina
oxide, clay), organic support(starch).
 Bonds of low energy are involved e.g. ionic interactions, hydrogen bonds,
van der Waals force, etc.
 If the enzymes is immobilized externally the carrier particle size must be
very small in order to achieve an appreciable surface of bonding.
There are four procedures for immobilization by adsorption
 (1) Static process (enzyme is immobilized on the carrier simply by
allowing the solution containing the enzyme to contact the carrier
without stirring )

10.  (2) Dynamic batch process (carrier is placed into the enzyme
solution and mixed by stirring or agitated continuously in
shaker)
 (3) The reactor loading process( carrier is placed into the
reactor that will be subsequently employed for processing
then enzyme solution is transferred to the reactor and
carrier is loaded in dynamic environment by agitating the
carrier and enzyme solution)
 (4) The elelctrodeposition process ( carrier is placed
proximal to one of the electrodes in an enzyme bath, the
current put on the enzyme migrates to the carrier and
deposited on the surface)

11. Entrapment
 Enzymes can be physically entrapped inside a matrix (support) of
a water soluble polymer such as polyacrylamide types gels and
naturally derived gels e.g. Agar, gelatin, carrageenan, alginate etc.
 The form and nature of matrix are vary.
 Pore size of matrix should be adjusted to prevent the loss of
enzymes from the matrix due to excessive diffusion.
 There are several methods of enzyme entrapment
 (1) Inclusion in gels ( enzyme entrapped in gels)
 (2) Inclusion in fibers( enzyme entrapped in fiber format
 (3) Inclusion in microcapsule( enzyme entrapped in microcapsule
formed monomer mixtures such as polyamine and polybasic
chloride, polyphenol and polyisocynate)

13. Disadvantages
 Enzyme leakage into solution
 Diffusional limitation
 Reduced enzyme activity and stability
 Lack of control micro-environmental conditions.

14. Encapsulation
 Encapsulation is the enclosing of a droplet of solution
of capsule in a semi permeable membrane Capsule.
 The capsule is made up of cellulose nitrate and nylon.
 The method of encapsulation is cheap and simple but
effectiveness largely depends on the stability of
enzyme although the catalyst is very effectively
retained within the capsule.
 The technique is restricted to the medical science

15.  In this method a large quantity of enzyme is
immobilized but the biggest disadvantage is that only
small substrate molecule is utilized with the intact
membrane.

16. Covalent Binding
 Covalent bond is formed between the chemical group of enzyme and
chemical groups on surface of carrier.
 Covalent bonding is thus utilized under a broad range of pH, ionic
strength and other variable conditions.
 Immobilization steps are attachment of coupling agent followed by an
activation process, or attachment of a functional group and finally
attachment of the enzyme.
 Different types of carrier are used in immobilization such as
carbohydrates proteins and amine- bearing carriers, inorganic carriers
etc.
 Covalent attachment may be directed to a specific group (e. g. amine,
hydroxyl, tyrosyl etc.) on the surface of the enzyme.
 Hydroxyl and amino groups are the main groups of the enzymes with
which it forms bonds, whereas sulphydryl group least involved.

17.  Different methods of covalent bonding are
 (1) Diazoation( bonding between the amino group of support e.g.
aminobenzyl cellulose, aminosilinised porous
glass,aminoderivatives and a tyrosyl or histidyl group of enzyme)
 (2) Formation of peptide bond.
 (3)Group activation ( use of cyanogen bromide to a support
containing glycol group i.e. cellulose, sephadex, sepharose etc.)
 Polyfunctional reagents( use of a bifunctional or multifunctional
reagent e.g. glutaraldehyde which form bonding between the
amino group of the support and amino group of enzyme)
 The major problem with covalent bonding is that the enzyme may
be inactivated by bringing about changes in conformation when
undergoes reactions at active sites.

18. Covalent Binding
 The most widely used method for enzyme immobilization
 It is technically more complex
 It requires a variety of often expensive chemicals
 It is time-consuming
 But immobilized enzyme preparations are stable and leaching is
minimal
 Enzymes are immobilized by a suitable group in the surface:
 Hydroxyl groups in supports (e.g cellulose, dextran, agarose)
 Amino, carboxyl and sulfhydryl groups in amino acids

19. Cross- Linking or copolymerization
 Cross- linking is characterized by covalent bonding
between the various molecule of an enzyme via poly
functional reagent such as glutaraldehyde