Immobilization of enzymes

B.Sc. III Microbiology
Syllabus Solapur University

1. Immobilization of Enzymes Methods and Applications Paper XII Unit II. Class- B.Sc.III Mr. Kamble Sainath Hanmant Assistant Professor in Microbiology D.B.F.Dayanand College of Arts and Science, Solapur

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

8. 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 )

9. (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)

10. 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)

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

12. 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

13. 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.

14. 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.

15. 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.

16. 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

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

18. Thank You