Enzyme Immobilisation

Enzyme Immobilisation

1. Explanation

   • (1) Immobilisation of enzymes involves a technique whereby an enzyme is bound immovably to an inert, insoluble material and so cannot dissolve in a solution containing its substrate.
   • (2) Therefore the enzymes can be held in place during the reaction and can also be reused.
   • (3) the enzyme molecules are held on (physically or chemically) or inside an insoluble support material, such as fibres, gels or plastic beads.
   • (4) They are then tightly packed into a column (continuous flow reactor) through which a supply of the reactants is continually passed to form the product.

2. What is an immobilised enzyme?

   • An immobilised enzyme is an enzyme that is attached (physically or chemically) to an inert, insoluble material.

3. Flow Rate:imoobilised enzymes.

   • the flow rate through the column influences activity.
   • if too slow ... the reaction is completed early in the column - less effective
   • if too fast ... not all the substance will be engaged in the reaction

4. methods of immobilising enzymes

   • Physical
     • ADSORPTION - the enzyme is attached to the outside of an inert material such as glass, alginate beads or a matrix
     • ENTRAPMENT - inside insoluble beads (alginate beads)
     • ENCAPSULATION - inside selectively permeable membrane
   • Chemical
     • CROSS LINKAGE - enzyme covalently bonded to a matrix by a chemical reaction

5. Using immobilised enzymes can reduce the rate of reaction as some of the active sites may be blocked.

6. Physical bonding generally results in lower enzyme activity than chemical bonding

7. Properties of material used for enzyme immobilisation

   • must be inert
   • must be permeable to allow substance and product to pass through it if entrapment is being used.
   • must be impermeable to enzyme if entrapment is being used
   • insoluble in nature so that the enzyme can be recovered

8. To increase efficiency of immobilised enzymes

   • reduce rate that substrate passes across the enzyme
   • Increase the enzyme concentration on the beads
   • Reduce the size of the enzyme rich beads (increases the surface area to volume ratio of the beads making it more likely that the enzyme will be on the outwards of the bead.

9. ADVANTAGES

   • The product is enzyme free
   • the enzymes can be reused - no purification process
   • enzyme is thermo stable - this means it remains active over a greater range of pH and temperature
   • can be continuous (FLOW)

10. DISADVANTAGES

    • The active site of the immobilised enzyme may be blocked by the support matrix - active site inaccessible
    • Slows down rate of reaction
    • amount of product produced per minute is reduced