Applied Enzymes Catalysis-3

1. Applied Enzymes Catalysis-3 Dr. A.K.M. Shafiqul Islam School of Bioprocess Engineering 19.01.10

2. Immobilization • Why we need Immobilization? • In actual practice, the soluble enzymes engaged in ‘batch operations’ is found to be not-so-economical due to the fact that the active enzyme is virtually lost (not recovered) after each viable reaction.

3. Immobilization • Therefore, in order to combat and overcome such a non-productive, economically not feasible, and deleterious effect the enzymes have been ultimately immobilized ; • and the process is termed as enzyme immobilization.

4. Immobilization • Immobilization is defined as — ‘confining the enzyme molecules to a distinct phase from the one wherein the substrates and the products are present’.

5. Immobilization • Immobilization of an enzyme means that it has been confined or localized so that it can be reused continuously

6. Important Features • Enzymes are more or less physically confined in the course of a definite continuous catalytic process. They may be suitably recovered from the reaction mixture and used over and over again thereby gainfully improving the economic viability of the entire process. • It may be accomplished by fixing the enzyme molecules to or within certain appropriate subtance. • It should be absolutely critical that both the substrates and the products migrate quite freely in and out of the phase to which the specific enzyme molecules are actually confined. • Certain enzymes which are as such readily inactivated by heat (i.e., thermolabile), may be rendered heat-stable by attachment to inert polymeric supports.

7. Important Features • Immobilized enzymes may be recycled, rapidly controlled, operated continuously, product(s) easily separable, and above all the enzymatic properties (i.e., stability, activity) altered favourably. • Immobilized enzymes seem to be fairly compatible with multienzyme procedures that essentially make use of enzyme-based cofactors. • Utilization of whole beads are invariably employed for a wide spectrum of catalytic functions thereby ascertaining the fact that immobilized enzymes do have an edge over their soluble counterparts overwhelmingly.

8. Reason for immobilization • For processing with isolated enzymes, • an immobilized form can be retained in the reactor • In solution, some enzymes will leave the reactor with the final product. • New enzymes be introduced to replace the lost ones, • Enzymes in the product may be undesirable impurities which must be removed. • Immobilized enzymes may retain their activity longer than those in solution • An immobilized enzyme may be fixed in position near other enzymes participating in a catalytic sequence, • thereby increase the catalyst efficiency for the multistep conversion.

9. Immobilized Enzyme Systems • Advantages of immobilization • Enzyme reutilization • Elimination of enzyme recovery and purification • May provide better environment for activity • Methods of immobilization • Surface immobilization • Adsorption • covalent binding • Ionic bonding • Entrapment • Encapsulation

10. Physical Method Where weaker interactions or containment of the enzymes are involved. • Enzymes may be adsorbed on a variety of carriers offering in some cases the practical convenience of simple regeneration by removal of deactivated enzyme and reloading with fresh, active catalyst. • If a support or entrapping material is used, its properties combined with those of the enzyme and the immobilization procedure dictate overall catalyst properties.

11. Chemical Method Where covalent bonds are formed with the enzyme • If the native surface is not ideal, can it be chemically modified or coated to facilitate enzyme attachment. • These materials have been employed for covalent enzyme immobilization • They have some interesting surface functional groups. • materials which have been used as immobilized enzyme supports include ceramics, glass and other metal oxides.

12. Immobilization Process • chemical methods • where covalent bonds are formed with the enzyme • physical methods • where weaker interactions or containment of the enzymes are involved

13. Enzyme immobilization • Four types of immobilization • Adsorption Method • Covalent bonding • Entrapment • Encapsulation

14. Adsorption Method • By allowing the contact of the enzyme and the polymer support either by percolating the enzyme via a packed bed, tube, membrane formed from a support material or in a stirred bioreactor. Percolating - Filtering especially through small holes or crevices.

15. Adsorption Method • The enzyme molecules get adhered to the surface of a carrier matrix on account of the spectacular combination of hydrophobic effects and the critical formation of several salt-linkages per enzyme molecule

16. Adsorption Method

17. Covalent Bonding • The covalent bonding of an enzyme may be accomplished either by activating the polymer with a reactive moiety (i.e., copolymerization with ethylene, anhydride of maleic acid) • or by effectively employing the bifunctional reagent to serve as a bridge between the two entities : enzyme and polymer

18. Covalent Bonding

19. Covalent Bonding • In such case, the enzyme invariably may get inactivated because the reactions normally engage a functional moiety strategically located at the ‘active site’ of the enzyme. • Thus, the overall net effect being the substantial loss of enzymatic activity. • The loss in enzymatic activity may be overcome by carrying out the ‘enzyme immobilization’ either in the presence of a competitive inhibitor or an enzyme substrate.

20. Insoluble materials and their surface functional groups useful for covalent enzyme attachment

22. Advantages of Covalent Bonding • Adsorption of enzymes to the carrier matrices is quite easy and convenient, and hence used extensively. • Covelent bonding attachment is not reversed by pH, ionic strength or substrate. • Relatively broader spectrum of bonding reactions, and of matrices with functional group capable of either having covalent bondage or prone to be activated to yield such groups renders this method into a highly acceptable one.

23. Interactions and carriers used for enzyme immobilization by adsorption CM = carboxymethyl; DEAF = diethylaminoethyl.

24. Advantage of Immobilized Enzymes • Enzyme in solution can be used once • It can be fixed on a carrier so can be used continuously • It can be bound, adsorbed, entrapped or crosslinked • They are more heat stable, pH is shifted

25. Thanks