1. Immobilization
• Immobilized biocatalysts can be made from
killed cells, cell fragments, purified
enzymes, and respiring cells
• Definition: Enzymes or cells which are
physically confined to a defined region in
space while retaining their catalytic activity
and have the ability to be repeatedly and
continuously used
2. Immobilized Enzyme Systems
Enzyme Immobilization:
- Easy separation from reaction mixture, providing the ability to
control reaction times and minimize the enzymes lost in the
product.
- Re-use of enzymes for many reaction cycles, lowering the total
production cost of enzyme mediated reactions.
- Ability of enzymes to provide pure products.
- Possible provision of a better environment for enzyme activity
- Diffusional limitation
3. • Higher dilution rates can be used in a
CSTR.
• Easier product recovery.
• Lowered viscosity.
• Easier enzyme recovery.
Why….
Advantages of Immobilization
4. • Mass transfer is inhibited
• Reduced activity of enzymes
• Bursting of immobilization medium
due to cell growth
• Cells/enzymes can leak out of
immobilization medium
Disadvantages of Immobilization
7. Membrane Microencapsulation
Membrane polymerized around aqueous
enzyme solution in colloidal suspension
(particle sizes on the order of 100-10 µm)
Organic solvent
Aqueous enzyme solution
mixing Add polymer
9. Other Membrane Immobilization
Methods- HFM
Hollow fiber reactors use micro- or ultra-
filtration membranes to retain high MW
enzymes, but pass low MW compounds
Enzymes
or cells
Substrate and
products outside
the membrane
Substrate
diffuses through
membrane
Product
diffuses out of
the membrane
14. Entrapment Immobilization is based on the
localization of an enzyme within the lattice
of a polymer matrix or membrane.
- retain enzyme
- allow the penetration of substrate.
It can be classified into matrix and micro
capsule types.
Immobilized Enzyme Systems
16. Entrapment
• Advantages
– Relatively benign attachment method
– Easy to perform
• Problems
– Mass transfer limitations
– Cell/enzyme leakage
– Some cell/enzyme deactivation
• Alginate (polymer) common
entrapment matrix
17. Immobilized Enzyme Systems
Matrix Materials:
Organics: polysaccharides, proteins, carbon, vinyl and allyl
polymers, and polyamides. e.g. Ca-alginate, agar,
K-carrageenin, collagen
Immobilization procedures:
Enzyme + polymer solution → polymerization
→ extrusion/shape the particles
Inorganics: activated carbon, porous ceramic.
Shapes: particle, membrane, fiber
18. Immobilized Enzyme Systems
Entrapment
challenges:
- enzyme leakage into solution
- diffusional limitation
- reduced enzyme activity and stability
- lack of control micro-environmental
conditions.
It could be improved by modifying matrix or
membrane.
20. Immobilized Enzyme Systems
Surface immobilization
According to the binding mode of the enzyme, this method
can be further sub-classified into:
- Physical Adsorption: Van der Waals
Carriers: silica, carbon nanotube, cellulose, etc.
Easily desorbed, simple and cheap,
enzyme activity unaffected.
- Ionic Binding: ionic bonds
Similar to physical adsorption.
Carriers: polysaccharides and synthetic polymers
having ion-exchange centers.
21. Immobilized Enzyme Systems
Surface immobilization
- Covalent Binding: covalent bonds
Carriers: polymers contain amino, carboxyl, sulfhydryl,
hydroxyl, or phenolic groups.
- Loss of enzyme activity
- Strong binding of enzymes
22. Covalent Bonding
• Most extensively used method of
immobilization due to high bond
strength
• Three elements- structural polymer,
enzyme molecule and bridge molecule
–Activity of an immobilized enzyme
is a strong function of the
hydrophilicity of the structural
polymer
23. Covalent Bonding To Support
Bridging molecule needs to be small, have two
reactive groups, and not bond at the active site
24. Immobilized Enzyme Systems
Cross-linking is to cross link enzyme
molecules with each other using agents such as
glutaraldehyde.
Features: similar to covalent binding.
Several methods are combined.
25. Copolymerization of Enzymes
• Copolymerization is performed with
multifunctional bridge molecules to yield
and insoluble product
• Usually an inert protein is also included
28. Immobilization by Adsorption
• Binding forces are ionic, hydrophobic,
hydrogen bonds, or Van der Waals’
interactions
• Binding is simple (stir together in a
beaker) but is reversible. Substrate
addition can cause desorption.
