Immobilization techniques

Immobilization techniques are used by the movement of cells and enzymes. First enzyme immobilized in Japan. The immobilized enzyme is aminoacylase. This process is carried out by various methods such adsorption, covalent bonding, cross linking, entrapment, encapsulation. Immobilization of cells is an alternative method for immobilization of enzymes.

1. Immobilization techniques (enzymes &cells) P. Saranya

2. Introduction 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. The process whereby the movement of enzymes, cells, organelles, etc., in space is completely or severely restricted usually resulting in a water-soluble form of the enzyme. An immobilized enzyme in one whose movement in space has been restricted either completely or to a small limited region .

3. Features of enzyme immobilization The enzyme phase is called as carrier phase which is water insoluble but a hydrophilic porous polymeric matrix. E.g. agarose , cellulose, etc. Enzyme phase may be in the fine particulate, membranous, or microcapsule. The enzyme in turn may be bound to another enzyme via cross linking. The support or carrier utilized in immobilization technique is not stable at particular pH, ionic strength, or solvent conditions. Hence, may be disrupted or dissolved releasing the enzyme component after the reaction.

4. Advantages of enzyme immobilization: Multiple or repetitive use of single batch of enzymes. Immobilized enzymes are usually more stable. Product is not contaminated with the enzyme. Easy separation of the enzyme from the product. Allows development of a enzyme reaction system. Reduces effluent disposal. Less labour input. Saving in capital cost. High enzyme substrate ratio .

5. Disadvantages of enzyme immobilization: Uses in industrial application are limited. Loss of catalytic properties in some enzymes. Some enzymes become unstable. Enzymes are inactivated by heat generated in the system. High cost for isolation, purification and recovery of active enzyme .

6. Adsorption: Adsorption involves physical binding of the enzymes or cells on the surface of an inert support. The support materials may be used organic and inorganic compound. e.g. (alumina, silica gel, calcium phosphate gel, glass) inorganic (starch, carboxymethyl cellulose, DEAE-cellulose, DEAE- sephadex ) organic. It involves weak forces such as van der Waals forces and hydrogen bonds. Absorbed molecules can be easily removed by minor chages in pH, ionic strength or temperature.

7. Methods: Static process : Immobilization to carrier by allowing the solution containing enzyme to contact the carrier.(with out stirring) Dyanmic process : Carrier is placed in the enzyme solution and mixed by stirring or agitation. Reactor loading process : Carrier is placed in the reactor, the enzyme solution is transferred to reactor. Electrode deposition : carrier is placed proximal to an electrode in an enzyme bath and the electricity is put on, the enzyme migrates to the carrier and deposited on the surface .

8. Advantages and disadvantages: Advantages : 1. Easy to carry out 2. No reagents are required 3. Minimum activation steps involved 4. Less disruptive to protein than chemical methods Disadvantages: 1. Desorption of enzymes from the carrier 2. Efficiency is less

9. Covalent binding: Enzymes are immobilized by the creation of covalent bonds between the chemical groups of enzymes and the chemical groups of the support. This is widely used technique. However, covalent binding is often associated with loss of some enzyme activity. It contain 4 methods: 1. C yanogen bromide activation 2. Diazotation 3. Peptide bond formation 4. Polyfunctional-reagents

10. Methods: Cyanogen bromide activation : The inert support materials (cellulose, sepharose, sephadox) containing glycol groups are activated by CNBr , which the binds to enzymes and immobilize. Diazotation: Bonding b/w two amine group. (amino benzyl cellulose, amino derivatives of polystyrene, aminosilanzed porous glass Peptide bond formation : Enzyme immobilization can also done by the formation of peptide bond b/w the amino groups of the support and the carboxyl groups of enzymes. Polyfunctional-agents : Some of the reagents such as glutaraldehyde can be used to create bond b/w amino groups of enzyme and amino group of support. (e.g. aminoethyl cellulose, albumin, amino alkylated porous glass)

11. Advantages and disadvantages: Advantages: 1. Strong linkage of enzyme to the support 2. No leakage or desorption problem 3. A variety of support with different functional groups available 4. Widely applicable Disadvantages: 1. Chemical modification of enzyme leading to functional conformation loss 2. Enzyme inactivation by changes in the conformation

12. Cross linking (copolymerization): The crossing linking between enzyme and carrier molecules via polyfuctional agents such as glutaraldehyde , diazonium salt, hexamethylene isocyanate .

13. Advantages and disadvantages: Advantages: 1. It is simple and cheap method. 2. Commonly used polyfunctional reagents. 3. This method used as commercially. Disadvantages: 1. Polyfunctional reagents can denature the enzyme.

14. Entrapment : Enzymes are physically entrapped inside a matrix. Bonds involved may be covalent or non-covalent bond. Matrix used will be water soluble polymer. Pore size of matrix is adjusted to prevent loss of enzyme. It contain three methods. 1. Inclusion in to the gels 2. Inclusion in fibres 3. Inclusion in microcapsules

15. Methods: 1. Inclusion in the gels : Enzymes are entrapped in the gels. 2. Inclusion in fibres : Enzymes are supported on the fiber format. 3. Inclusion in microcapsules : Enzymes are entrapped in microcapsules formed by monomer mixtures such as polyamine, calcium alginate.

16. Advantages and disadvantages: Advantages : It is fast and cheap method. Mild conditions are required. Less chance of conformational changes in enzyme. Disadvantages : Leakage of enzyme. Pore diffusion limitation. Chance of microbial contamination.

17. Encapsulation: Encapsulation is a type of entrapment. It refers to the process of spherical particle formation where in a liquid or suspension is enclosed in a semi-permeable membrane. The membrane may be polymeric, lipoidal , lipoprotein based on non-ionic in nature. This method is recently being used in mammalian cells. For instance, pancreatic cells grown in cultures can be immoboilized by encapsulation. Hybridoma cells have also been immobilized successfully by this technique.

18. Advantages and disadvantages: Advantages: It is simple and cheapest method. Large quantity of enzymes can be immobilized by encapsulation. Disadvantages: Pore size limitation. Only small substrate molecules is able to cross the membrane .

19. Immobilized of cells: It is an alternative to enzyme immobilization. Well developed method for the utilization of enzymes from microbes. Effective method when: Individual enzymes become inactive during immobilization. Isolation and purification of enzyme is not cost effective. Methods of cell immobilization are same as described for enzyme immobilization. Immobilized of cells have been used for the treatment of sewage.

20. Immobilized viable cells: The viability of the cells can be preserved by mild immobilization. Some immobilized cells are particularly useful for fermentations. Sometimes mammalian cell cultures are made to function as immobilized viable cells.

21. Immobilized Non-viable cells: Immobilized non-viable cells are preferred over the enzymes or even the viable cells. The costly isolation and purification process. The best example is the immobilization of cells containing glucose, isomerase for the industrial production of high fructose syrup.

22. Advantages and disadvantages: Advantages: 1 . Multiple enzymes can be introduced to single step. 2. Enzymes are stable for long time. 3. Native conformation of enzyme is best maintained. 4. Cell organelles like mitochondrial and chloroplasts can be immobilized. Disadvantages : 1. Concentration of enzymes will be less. 2. Production of unwanted enzymes and unwanted products. 3. Modification of end products by other enzymes.

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