1. Immobilization of enzymes
Dr. Bhavesh Patel
Principal
V.P. and R.P.T.P. Science College, Vallabh Vidyanagar -

2. Immobilization of enzymes
Enzyme immobilization can be defined as restricting the enzyme molecule in a distinct phase from the one in which substrates and products are present.
The materials used for immobilization of enzyme are called Carrier matrices, this are usually inert polymers or inorganic materials.

3. Ideal carrier matrix Properties :- low cost inert stable
physical strength
enhancement of enzyme specificity
have regenerability after use
reduction in product inhibition
reduction in microbial contamination
nonspecific adsorption

4. Methods for immobilization
a) Adsorption b) Covalent binding

5. Adsorption
The enzyme here are adhere to carrier matrix due to hydrophobic effects or by forming salt links.
Binding is strong but may be weakened by substrate, pH etc.
Commonly used matrices :-
ion exchange matrices, porous carbon,clays, hydrous metal oxides, polymeric aromatic resins etc.

6. Covalent binding
The enzyme here are attached to certain matrix by forming covalent bonds
Binding is very strong. It generally occurs with side chain of the enzyme. Lysine is very useful, as they are very reactive, usually exposed to surface and very rarely occur at active site of enzyme.
Commonly used matrices :-
agarose, cellulose, poly acrylamides etc.

7. Cont….
Activation by CNBr

8. Cont….
Activation by Ethyl chloroformate

9. Cont….
Immobilization of enzyme using Glutaraldehyde

10. Methods for immobilization
c) Entrapment d) Membrane confinement

11. Entrapment
The enzyme here are entrapped with in suitable gels or fibers, with or with out covalent bonds.
Commonly used matrices :-
Cellulose acetate fibers, calcium alginate etc.

12. Membrane confinement
The enzyme molecule here are confined to semipermiable membrane. Thus in a aqueous phase beg of membrane is free to move with out allowing enzyme to come out.

13. Advantages
Enzymes are costly, Immobilization permits their repeated use.
The product can be easily separated without any additional cost.
Immobilized enzymes can be used in nonaqueous system.
Continuous production system can be used.
Thermo stability of some enzyme can be increased by immobilization.
Enzyme can be used at much higher concentration then free enzyme.

14. Disadvantages Additional cost.
It sometime affects stability and activity of enzyme.
This approach can not be used when one of the substrates is insoluble.
Some immobilization methods restricts the diffusion of substrate.

15. Applications of immobilized enzymes
Enzyme Substrate Product
Glucose isomerase D-glucose HFCS
Invertase Sucrose Invert sugar
Lipase Vegetable oil Cocoa butter substitute
Lactase Milk and whey Lactose free milk &whey
Nitrile hydratase Acrylonitrile Acrylamide
Raffinase Raffinose Raffinose free solution
Glucoamylase Dextrins D-glucose