Immobilization of enzymes Dr. Bhavesh Patel Principal V.P. and R.P.T.P. Science College, Vallabh Vidyanagar Email- bhavesh1968@rediffmail.com

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.

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

Methods for immobilization a) Adsorption b) Covalent binding

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.

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.

Cont…. Activation by CNBr

Cont…. Activation by Ethyl chloroformate

Cont…. Immobilization of enzyme using Glutaraldehyde

Methods for immobilization c) Entrapment d) Membrane confinement

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.

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.

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.

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.

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