22. Immobilization

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22. Immobilization

• Immobilization, which restrict the freedom of
  movement of biocatalyst (enzymes or cells) and
  provides physical support for cells, can be
  achieved by several ways.

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Methods of Biocatalyst Immobilization
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Adsorption to insoluble organic and inorganic
support like cellulose, dextran, nylon and
bentonite is mediated by ionic, hydrophobic or
hydrogen bonds. Adsorption is easily achieved by
mixing together the biocatalyst and a support,
but also desorption occurs after the changes in
pH, substrate concentration and ionic strength.
Since existing surface chemistry between
biocatalyst and support is used, little damage to
cells and enzymes is done.
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• It's simple, cheap and quick to immobilize cells
  or enzymes with this method,
• It does not cause chemical changes on support or
  biocatalyst.
• Moreover, reversibility of adsorption reaction
  allows regeneration of support with enzyme or
  cell.
• However, there are some disadvantages like
  nonspecific binding, overloading on support and
  leakage of enzymes/cells which cause
  contamination of product.

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Covalent attachment
Covalent attachment to chemically activated
supports is the most widely used way of
immobilizing enzymes. It occurs via activation of
functional groups present on surface of support
by specific agent and then addition of enzyme to
form covalent bond with support.
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• As a result of activation, functional groups
  become electrophilic (electron deficient) and
  react with electron donating functional groups of
  amino acids on surface of enzyme. Amino (NH2)
  group of lysine or arginine, carboxyl (CO2H)
  group of aspartic acid and glutamic acid,
  hydroxyl (OH) group of serine and threonine are
  examples of amino acid functional groups
• Polysaccharide polymers cellulose, dextran,
  starch, agarose, porous silica, porous glass are
  examples to these supports.

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Encapsulation
Containment of biocatalyst behind a barrier is
called encapsulation. Synthetic membranes, which
allow free movement of substrate and products,
preventing leakage of enzymes and cells can be
made from nylon, cellulose nitrate etc. In
addition encapsulation in liposomes is possible.
There are some problems associated with
encapsulation, for example, if products
accumulate rapidly in membrane, they may cause
rapture of membrane.
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Entrapment
Cells or enzymes can be entrapped in synthetic
polymers during polymerization of monomers.
Calcium alginate, chitosan, cellulose acetate,
collagen, agarose, polyacrylamide can be used as
matrix for entrapment.
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Self aggregation
Self aggregation is joining of cells or enzyme
forming large complex structure, it can occur
naturally (mycelial pellets or microbial flocs)
or can be artificially induced and involve
covalent bond formation between cells by means of
multifunctional reagents (glutaraldehyde
crosslinking). CH2 (CH2CHO)2
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Advantages of immobilization

• Reuse or continuous use of biocatalyst is
  possible,
• Biocatalyst does not contaminate the product,
• Substrate is equally supplied to each immobilized
  enzyme or cell,
• Generally stability of biocatalyst increased,
• Immobilised enzymes permit the use of enzymes
  from organisms which would not normally be
  regarded as safe
• Multienzyme systems can be developed

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Disadvantages of immobilization

• Enzyme activity may be lost by immobilization
• Extra cost
• Biocatalyst bound to solid support occupies
  larger volume in fermenter.

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PRACTICAL APPLICATION OF IMMOBILIZED BIOLOGICAL
CATALYST SYSTEMS

• The production of 6-amino penicillanic acid
  (6-APA) for semi synthetic penicillin production.
  
• Whole milk lactose hydrolysis is carried out by
  fiber entrapped lactase.
• Saccharification of starch by immobilized
  glucoamylase
• Cheese whey lactose hydrolysis by bound
  B-galactosidase

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• The most widely employed use of immobilized cells
  however are glucose isomerization and the
  hydrolysis of raffinose in beet sugar using
  mycelial pellets of the fungus Mortierrella sp.
• Raffinose (in beet molasses) is hydrolyzed to
  sucrose and galactose by Bgalactosidase
  (mellibiase) produced by the fungus.