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1. ENZYME AND CELL IMMOBILIZATION

2. Definition of Enzyme/Cell Immobilization The process where imprisonment of enzyme or cell is done in a distinct support or matrix due to which movement of enzymes in space is completely or severely restricted. Support or matrix allows exchange of medium containing substrate. FACTS Immobilized enzymes/ cells are also sometimes referred to as insolubilized, supported or matrix-linked enzymes/cells.

3. Need of Enzyme Immobilization- 1.Economic Nature- In most of the processes, enzymes are mixed in a solution with substrates and cannot be economically recovered after the reaction and are generally wasted. This problem can be overcome by using enzymes in an immobilized or insolubilized form so that they can be retained in a biochemical reactor for further catalysis. 2. Increased Stability- Immobilized enzymes typically have greater thermal and operational stability than the soluble form of the enzyme. Enzymes are fixed to an inert carrier in most stable functional form and sites for sensing the variation in temperature and pH are blocked by immobilization.  

4. Need for immobilizing cells Immobilized enzymes are not suitable for multienzyme reactions. The whole cells or cellular organelles can be immobilized to serve as multi-enzyme systems. Extracted intracellular enzymes are unstable. So, whole cell is immobilized.

5. Materials used for fabrication of immobilization supports

6.   TECHNIQUE OF ENZYME/CELL IMMOBILIZATION :- The techniques employed for immobilization of cells are almost the same as that used for immobilization of enzymes 1 .       Carrier binding.    Physical adsorption. Covalent bonding. Ionic bonding. 2.       Cross linking 3.       Entrapment Inclusion in- gel ; fiber ; microcapsules Microencapsulation.

7. 1. Carrier Binding: a) Physical Adsorption This method is based on the physical adsorption of enzyme protein on the surface of water-insoluble carriers like silica gel, calcium phosphate gel, glass, starch, DEAE cellulose. The bond between the enzyme and carrier molecule may be ionic, covalent, hydrogen, coordinated covalent or even combination of any of these.

8. Coupling of enzyme can be done either to external or internal surface of the carrier. External surface binding method is advantageous as it does not involve conditions like pore diffusion. The disadvantages include exposure of enzymes to microbial attack, physical abrasion of enzyme due to turbulence associated with solution. Internal Surface major disadvantage is the pore diffusion . Adsorbed enzymes can be easily removed by minor changes in pH, ionic strength or temperature. This is a disadvantage for industrial use of enzymes.

9. Methods of immobilization by physical adsorption- 1.      Static Process:-   In this technique, enzyme is immobilized by allowing it to be in contact with the carrier without agitation. 2.      Dynamic Process:-  This process typically involves the mixing of enzyme with the carrier under constant agitation using mechanical shaker. 3.      Reactor loading:-  This process is employed for commercial production of immobilized enzymes. Carrier is placed in reactor and enzyme solution is transferred to reactor with agitation of the whole content in the reactor. 4.      Electro-Deposition:- Carrier is placed in vicinity of one of the electrode in an enzyme bath and electric current is applied leading to migration of enzyme towards the carrier. This results in deposition of enzyme on the surface of the carrier

10. 1. Carrier Binding: b ) Covalent Bonding Immobilization of the enzymes can be achieved by creation of covalent bonds between the chemical groups of enzymes and the chemical groups of the support. However, covalent binding is often associated with loss of some enzyme activity. The inert support usually requires pretreatment (to form pre-activated support) before it binds to enzyme.

11. Methods of Covalent Binding 1. Cyanogen bromide activation: The inert support materials (cellulose, sepharose, sephadex) containing glycol groups are activated by CNBr ( Cyanogen Bromide) which bind to enzymes. 2. Diazotation : Some support materials (amino benzyl cellulose, amino derivatives of polystyrene) are subjected to diazotation on treatment with NaNO 2  and HCI. They, in turn, bind covalently to tyrosyl or histidyl groups of enzymes. 3. Peptide bond formation: Formation of peptide bonds between the amino (or carboxyl) groups of the support and the carboxyl (or amino) groups of enzymes. 4. Activation by bi- or poly-functional reagents: Some of the reagents such as glutaraldehyde can be used to create bonds between amino groups of enzymes and amino groups of support ( aminoethylcellulose , albumin, amino alkylated porous glass).

12. 2.CROSS LINKING The absence of a solid support is a characteristic feature of immobilization of enzymes by cross- linking. The enzyme molecules are immobilized by creating cross-links between them, through the involvement of poly-functional reagents. Reagents react with the enzyme molecules and create bridges which form the backbone to hold enzyme molecules. Reagents include-glutaraldehyde, diazobenzidine , hexamethylene diisocyanate .

13. Glutaraldehyde is the most extensively used cross-linking reagent. It reacts with lysyl residues of the enzymes and forms a Schiff’s base. The cross links formed between the enzyme and glutaraldehyde are irreversible and can withstand extreme pH and temperature. Eg. glucose isomerase, penicillin amidase are immmobilized by glutaraldehyde Advantage- The technique of cross-linking is quite simple and cost-effective. Disadvantage- It involves the risk of denaturation of the enzyme by the poly-functional reagent.

14. 3. Entrapment In entrapment, the enzymes or cells are not directly attached to the support surface, but simply trapped inside the polymer matrix. The size of the matrix pores is such that the enzyme is retained while the substrate and product molecules pass through but disadvantage is that low molecular weight enzymes are lost In this technique the enzyme is not subjected to strong binding forces and structural distortions but some deactivation may occur due to changes in pH or temperature or addition of solvents. The matrices used- polyacrylamide gel, collagen, gelatin, starch, cellulose, silicone and rubber. Enzymes can be entrapped by several ways.

15. a. Methods of Entrapment Inclusion in Gels- Enzymes trapped inside the gels. Inclusion in Fibers- Enzymes supported on fibers made of matrix material. Inclusion of Microcapsules- Enzymes are trapped inside a microcapsule matrix formed by monomer mixture such as polyamine and calcium alginate.

16. b. Microencapsulation- It is type of entrapment . It is done by enclosing the enzymes in a spherical membrane capsule. Capsule will be made of semi permeable membrane that is impermeable to enzymes and other molecules but is permeable to substrate and product. like nitro cellulose or nylon. Effectiveness depends on stability of enzymes inside capsule.

17. Advantages of Enzyme 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

18. Disadvantages of Enzyme Immobilization It gives rise to an additional bearing on cost for purification and recovery of enzyme. It affects the stability and activity of enzymes. The technique may not prove to be of any advantage when one of the substrate is found to be insoluble. Catalytic properties of some enzymes are reduced or completely lost after immobilization. Industrial applications are limited. Enzymes are inactivated by heat generated in system.

19. Example of Immobilized Cells with methods-