Immobalization

1. Immobilization of
Enzymes

2. Introduction
 Definition
 Used in various industrial processes
 reversible physical adsorption and ionic linkages to stable
covalent bonds
 Can alter catalytic activity
 Thermal stability can alter

3. Background
 What are enzymes?
 Functions
 Immobilization of enzymes
 1967 by Chibata - immobilization of Aspergillus oryzae
 Used in biotechnological product

4. Background

5. Background

6. History

7. Choice of Supports
Ideal support should have-
 Physical resistance to compression,
 Hydrophilicity
 Inertness toward enzymes
 Ease of derivatization
 Biocompatibility
 Resistance to microbial attack
 Availability at low cost

8. Choice of Support

9. Choice of Supports
 Physical characteristics
 Porous nature
 Hydrophilic
 Agarose

10. Choice of Support

11. Irreversible immobilization
Covalent Binding
 Creation of covalent bonds between chemical groups of
enzymes and support
 Loss of some enzyme activity
 Covalent coupling

12. Irreversible immobilization
Covalent Binding
Cynogen bromide activation

13. Irreversible immobilization
Covalent Binding
Gluteralehyde

14. Irreversible immobilization
Covalent Binding
Diazotization
Peptide bond formation
-R-NH2
NaNO2
HCl
-R-N+
= HCl-
Enzymes
-R-N=N- Enzymes
-R-COOH CH3OH
HCl
-R-COOCH3
Hydrazine -R-CONH-NH2
NaNO2, HCl
-R-CONH-Enzyme
Enzymes

15. Irreversible immobilization
Entrapment
 entrapment of enzymes
Three dimensional gel
In situ polymerization
Polycondensation – with polyalcohols, polyamines
 Inclusion in gel
 Inclusion in fibers

16. Irreversible immobilization
Microencapsulation
 Membrane reactors
 Emulsions
 Microcapsules
 Dehydrated rehydrated vesicles
 Reverse phase osmosis method

17. Reversible immobilization
Microencapsulation
 Membrane reactors
 Emulsions
 Microcapsules
 Dehydrated rehydrated vesicles
 Reverse phase osmosis method

18. Reversible immobilization
Adsorption (Noncovalent Interactions)
 Nonspecific Adsorption
 Ionic binding
 Hydrophobic Adsorption
 Affinity binding

19. Reversible immobilization
Adsorption (Non covalent Interactions)
 External or internal adsorption
 Methods
– Static pores
– Dynamic pores
– Reactor loading
– Electro deposition

20. Reversible immobilization
Complexation and chelation
 Metal link chelation
Zirconium or titanium
 Chloroquo complexes of titanium in HCL solution

21. Stabilization of enzymes
 Solvent stabilization
 Substrate stabilization
 Stabilization by polymers
 Stabilization by salts
 Stabilization by chemical modifications
 Stabilization by rebuilding