Immobilization

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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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Advantages of Immobilization

• Higher dilution rates can be used in a CSTR.
• Easier product recovery.
• Lowered viscosity.
• Easier enzyme recovery.
• Why.

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Disadvantages of Immobilization

• Mass transfer is inhibited
• Reduced activity of enzymes
• Bursting of immobilization medium due to cell
  growth
• Cells/enzymes can leak out of immobilization
  medium

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Immobilization Methods
Physical
Chemical
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Membranes
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Membrane Microencapsulation

• Membrane polymerized around aqueous enzyme
  solution in colloidal suspension (particle sizes
  on the order of 100-10 µm)

Add polymer
mixing
Organic solvent
Aqueous enzyme solution
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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

Substrate and products outside the membrane
Product diffuses out of the membrane
Enzymes or cells
Substrate diffuses through membrane
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Close-up view of fiber
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Hollow Fiber Reactor
Enzymes or cells in
Product out
Substrate in
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Two Types of Cartridges
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Entrapment
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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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Covalent Bonding
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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 Support

• Bridging molecule needs to be small, have two
  reactive groups, and not bond at the active site

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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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Adsorption
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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