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Immobilization

Immobilization. Immobilized biocatalysts can be made from killed cells, cell fragments, purified enzymes, and respiring cells

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Immobilization

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

  3. 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

  4. Immobilization Methods Physical Chemical

  5. Membranes

  6. 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

  7. Microencapsulation • Advantages • High surface to volume ratio • Thin membrane • Relatively benign attachment method • Problems • More easily damaged than gel beads

  8. 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

  9. Close-up view of fiber

  10. Hollow Fiber Reactor Enzymes or cells in Product out Substrate in

  11. Two Types of Cartridges

  12. Entrapment

  13. 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

  14. Covalent Bonding

  15. 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

  16. Covalent Bonding To Support Bridging molecule needs to be small, have two reactive groups, and not bond at the active site

  17. Copolymerization of Enzymes • Copolymerization is performed with multifunctional bridge molecules to yield and insoluble product • Usually an inert protein is also included

  18. Chemistry of Covalent Immobilization

  19. Adsorption

  20. 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.

  21. Typical Adsorbents • Cellulose • Polystyrene resins • Kaolinite • Glass • Alumina • Silica gel

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