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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
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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
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• Higher dilution rates can be used in a CSTR.
• Easier product recovery. • Lowered viscosity.• Easier enzyme recovery.
Why….
Advantages of Immobilization
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• 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
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Immobilization Methods
Membranes Matrix entrapment Adsorption Covalent bonding
Immobilization
OrganicPolysaccharidesProtein chains
Synthetic Polymers
InorganicCeramic
Glass, SilicaAlumina
Supports
PhysicalChemical
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Membrane MicroencapsulationMembrane 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
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Microencapsulation
• Advantages
–High surface to volume ratio
–Thin membrane
–Relatively benign attachment method
• Problems
–More easily damaged than gel beads
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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
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Close-up view of fiber
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Hollow Fiber ReactorEnzymes or cells in
Substrate inProduct out
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Two Types of Cartridges
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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
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Immobilized Enzyme Systems
Entrapment - Matrix Entrapment - Membrane Entrapment
(microencapsulation)
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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
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Immobilized Enzyme SystemsMatrix 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
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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.
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Immobilized Enzyme SystemsSurface immobilization
According to the binding mode of the enzyme, this method can be further sub-classified into:
- Physical Adsorption: Van der WaalsCarriers: 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.
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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
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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
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Covalent Bonding To SupportBridging molecule needs to be small, have two reactive groups, and not bond at the active site
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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.
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Copolymerization of Enzymes• Copolymerization is performed with
multifunctional bridge molecules to yield and insoluble product
• Usually an inert protein is also included
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Chemistry of Covalent Immobilization
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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.
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Typical Adsorbents
• Cellulose
• Polystyrene resins
• Kaolinite
• Glass
• Alumina
• Silica gel