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Cell and Enzyme Immobilization - Jmdsdf

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									Cell and Enzyme Immobilization
Cells and enzymes as biocatalysts

             enzyme


         S            P


              cells
cell based versus enzymatic
         processes
     glucose                       glucose/fructose

                glucose isomerase




 glucose                                        ethanol
           multi-enzymes acting sequentially


  • whole cells preferred when multi-step
  • enzymes preferred for 1 or 2 step transformations
       • competing side reactions with whole cells
       • sterility problems
       • cell lysis
       • other physiological requirements (nutrients, O2)
     Advantages to immobilizing
        enzymes and cells
• increased stability,weeks or months
• stable to heat, pH extremes, storage,
  reaction
• facilitates recovery for repeated or
  continuous use (essential for soluble
  enzymes)
• cellular activity is enzymatic activity
  (biotransformations)
                Immobilization Techniques


    entrapped                     bound


matrix                 adsorbed             covalently
encapsulation                               attached

        microencapsulation            support   enzyme or
                                                cell
   Matrix or lattice entrapment in
           polymeric gels
• monomer, crosslinker,
  polymerization catalyst, cells or
  enzyme
• forms lattice structure, entrapping
  cells/enzyme
• eg. polyacrylamide cross-linked
  with N,N'-methylenebisacrylamide
  (covalent gel)
Alginate and carrageenan non-covalent gels


          • Naturally derived polymers extracted from
            seaweed
          • Used in food industry as a thickener
             – ice cream, pudding, frozen drink
               concentrates, jam, yoghurt, bakery
               products, confectionery
          • Dental molds
          • Immobilization technology as an
            encapsulating matrix
          • Natural polymers are highly variable in
            composition and their chemistry is generally
            not known
          • Composition affects properties
                               Alginate


             a-L-mannuronic acid                          b-D-guluronic acid




Alginate polymer



Alginate block
structures


      (Mikkelsen and Elgsaeter, 1995; Smidsrod and Skjak-Braek, 1990)
             Alginate Matrix

Binding of Ca2+ to G



Eggbox model for
Ca2+ binding



Structure of the
Alginate-Ca2+ Matrix
                Cell entrapment protocol
                  - external gelation




                            Ca++


Dropwise addition of
alginate/cells into CaCl2
gelation bath
DNA entrapment protocol
- emulsification/internal gelation

                                            Ca++   6.5   7.5   CaCO3




                       alginate droplet
                      containing DNA,
                   microcrystalline CaCO3

 alginate in oil
   emulsion
                                               oil recycle

canola oil: 40oC                       KCl




carrageenan: 40oC                            5oC

                              static mixer

                                       separator
                                          settler
yeast          static mixer
          o
        40 C
                                 carrageenan beads
                                    to bioreactor
                   Immobilized yeast
                      technology




Kenics static mixer to
encapsulate brewing
yeast



 Continuous brewing
gas out
              beer out

          bead disengagement
          section


                           Labatt continuous
          draft tube
                           airlift reactor

          temperature
          control jacket

      medium in

sparger - air in
Tannase from Aspergillus oryzae to
      hydrolyze tea tannins
• tannins represent 25% of extractables
      in tea leaves
• cause creaming (turbidity) on cooling
• desire tea to be clear and bright
• tannase controlled hydrolysis of tannins,
       retaining flavour
• encapsulated tannase remained stable
       for 1 month
• 3 successive batch cycles during
      48 h processing
Membrane coating

   polyanion core
   (alginate)

   polycation
   membrane
   • chitosan
   • poly-L-lysine
   • co-guanidine
DNA microspheres following GI transit
    Damon/Connaught process to
    encapsulate pancreatic islets



                coated with
                poly-L-lysine
islets in                       liquify alginate
alginate bead                   core with citrate
                                or EDTA
            Microencapsulation
• spherical ultrathin semi-
  permeable membrane
  enclosing cell/enzyme
  suspension/solution
• interfacial polymerization
  reaction (nylon)

NH2(CH2)6NH2 + ClCO(CH2)8COCl

      NH2(CH2)6NH-CO(CH2)8CONH(CH2)6NH-CO(CH2)8CO-     +   HCl

                                nylon 6-10 polyamide
Microencapsulation protocol
- interfacial polymerization

                               oil soluble
                               cross-linker
              chitosan




    cells/chitosan
    in oil emulsion
Encapsulation of lobster carotenoids as
natural food pigment
                Adsorption
• simple adsorption of
  cell/enzyme onto support
  (carrier) with adsorptive
  properties
  – anion exchange resins (DEAE
    cellulose, Sephadex)
  – cation exchange resins
    (carboxymethylcellulose)
   Covalent binding to support
• common technique
• carriers
  – natural materials (cellulose, active
    carbon)
  – inorganic materials (glass, stainless
    steel, ceramics (porous), silica (sand)
  – enzymes/cells have reactive groups
    (NH2, OH, SH, COOH)
  – carriers are usually unreactive so
    activation step required
     Corning glass process (glucose isomerase and lactase)



1. support activation

ceramic + (C2H5O)3Si(CH2)3NH2                      ceramic-Si-(C2H5O)2(CH2)3NH2

           (3-aminopropyltriethoxysilane)                       (activated support)




2. cross-linking of cells/enzyme

                 cells-NH2       +      OHC-(CH2)3-CHO

                                            ( glutaraldehyde)



  cell-N=CH(CH2)3CH=N-carrier + H2O
  Cross-linking intramolecular or cell to cell

• enzyme or cell cross-linked to
   – another enzyme molecule
   – another protein (BSA)
   – insoluble carrier molecule
• glutaraldehyde cross-links NH2 groups
• hexamethylene diamine links COOH groups
  Comparison of immobilization techniques

• adsorption and gel entrapment
   – simple, gentle and efficient
   – enzyme/cells often released (leaky); solved by cross-linking
   – gas buildup may be problem
• microencapsulation
   –   size exclusion (eg. antibodies)
   –   only small substrates can be used
   –   may lead to inactivation
• covalent attachment and cross-linking
   – strong attachment
   – laborious and expensive
   – often leads to significant inactivation
 Reaction kinetics or mass transfer control

• diffusional resistances           boundary film
  minimized by
  – decreasing particle
    size (increase surface                     Rbulk
    area/volume ratio)
  – increasing [R]bulk
  – improved mixing,
    agitation
  – increasing porosity
  – optimizing distribution
    of enzyme/cells
CH3CHOHCOOH + O2             CH3COOH + CO2 + H2O

         L-lactate-oxygen 2-oxidoreductase

								
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