Using immobilised enzymes

Isolated enzymes

• Clear advantages
  • less wasteful --> don't produce excess biomass
  • more efficient --> work at higher concentrations that if whole microorganisms were used
  • more specific --> no unwanted enzymes present
  • maximise efficiency --> can be given ideal conditions for maximum product formation
  • less downstream processing --> pure product is produced
• Most isolated enzymes are extracellular as they're generally easier and cheaper to use than intracellular 
  • they are secreted so easier to isolate
  • each microorganisms produces few extracellular enzymes so its easy to identify and isolate the desired one
  • they tend to be more robust that intracellular ones (adapted to cope in greater variations of pH and temperature)
• But intracellular enzymes are sometimes isolated and used because there's a wider range of them and in some cases they're the only ideal one
  • benefits of using very specific intracellular enzymes sometimes outweigh the smaller costs of using an extracellular one
    • glucose oxidase for food preservation
    • asparaginase for cancer treatment
    • penicillin acylase for converting natural penicillin to semi-synthetic drugs

Immobilised enzymes

• isolated enzymes are more efficient that using whole organisms but often very wasteful
• enzymes aren't cheap and at the end of process are easily lost
• increasingly, industries are using immobilised enzymes instead (attached to an inert support system which the substrate is passed over and the products are formed)
• because they are held stationary during the process they can recovered from the reaction mixture and reused --> they don't contaminate the end product so downstream processing is needed less
• Advantages
  • can be reused so are cheaper
  • easily separated from reactants so less downstream processing - cheaper
  • more reliable because there's a high degree of control over the process
  • they have a greater temperature tolerance as they're less easily denatured
  • easily manipulated so can keep bioreactors running continuously
• Disadvantages
  • reduced efficiency as the immoblising process may reduce their activity rate
  • higher initial costs of materials as immobilised enzymes are more expensive than free enzymes
  • higher initial costs of the bioreactor as they system is different
  • more technical issues as the systems are more complex

How are they immobilised?

• Adsorption to inorganic carriers (cellulose, silica) 
  • Adv = cheap, simple, used for many processes, enzymes very accessible
  • Disadv = enzymes can be lost from matrix easily
• Covalent or ionic bonding to inorganic carrier (covalent to carrier with amino group or ionic to polysaccharides)
  • Adv = cost varies, enzymes are strongly bound, very accessible to substrate, pH and substrate concentration have little effect on activity
  • Disadv = cost varies, active site may be modified in the process
• Entrapment in a matrix (polysaccharides, gelatin)
  • Adv = widely applicable to different processes
  • Disadv = may be expensive, can be difficult, diffusion to and from active site can be slow, effect on activity can vary
• Membrane entrapment in microcapsules (polymer-based semi-permeable membranes)
  • Adv = relatively simple, relatively small effect on activity, widely applicable
  • Disadv = relatively expensive, diffusion to and from active site can be slow

Uses of immobilised enzymes

• Penicillin acylase --> make semi-synthetic penicillins from natural penicillin to use against penicillin resistant strains or for people who're allergic to penicillin
• Glucose isomerase --> used to convert glucose to fructose as its sweeter so less needs to be used in foods to make them taste the same 
• Lactase --> used to make lactose-free dairy products for lactose intolerant people (converts lactose to glucose and galactose)
• Aminoacylase --> used to produce pure samples of L-amino acids
• Glucoamylase --> used to complete the breakdown of starch to glucose syrup