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Immobilized enzyme:
o Since enzymes are globular proteins and are soluble in water, so it is
difficult to separate enzymes for reuse in a batch process.
oEnzymes can be immobilized from surface or inside from a insoluble
matrix.
oFrom soluble form they can be immobilized by retaining them with a semi
permeable membrane.
oA main advantage of immobilized enzyme is that it can be reused since it
can easily be separated from the reaction solution and an be easily retained
in a continues flow reactor.
o Immobilized enzymes may show selectively altered chemical or physical
properties and it may stimulate the realistic natural environment where the
enzyme came from the cell.
Immobilization techniques:
Immobilization techniques can be classified by two
methods:
1.Chemical method (covalent bond formation
dependent)
2.Physical method (non-covalent bond formation
dependent)
1). Chemical method:
oA) Covalent attachment: the covalent attachment of enzyme
molecules via non-essential amino acids residue to water
insoluble, functionalized supports are the most widely used
methods for immobilizing enzymes.
oFunctional groups of the non essential amino acid residue that
are suitable for the immobilization process are free α-, β-, or y-
carboxyl groups, α- or β-amino groups, and phenyl, hydroxyl,
sulfhydryl, or imidazole groups.
B)Cross-linking Using Multifunctional Reagents:
o Water-insoluble enzymes can be prepared by using multifunctional
agents that are all bifunctional in nature and have low molecular
weight, such as glutaraldehyde.
o Enzymes can be reacted with multifunctional reagent alone so that
they are cross-linked intermolecularly by the reagent to form a
waterinsoluble derivative.
o Another method is to adsorb enzymes on a water-insoluble,
surface-active support followed by intermolecular cross-linking
with multifunctional reagents to strengthen the attachment.
o Multifunctional reagents can be also used to introduce functional
groups into water-insoluble polymers, which then react covalently
with water-soluble enzymes.
2). Physical methods:
oThis method is the simplest way to immobilize enzymes. Enzymes can
be adsorbed physically on a surface-active adsorbent by contacting an
aqueolls solution of enzyme with an adsorbent. Commonly employed
adsorbents are alumina, anion-exchange resins, calcium carbonate,
carbon, cationexchange resins, celluloses, clays, collagen, colloid-ion,
conditioned metal, glass plates, diatomaceous earth, and
hydroxyapatite.
Advantages & disadvantages of physical
methods
1. The procedure of immobilization is simple.
2. It is possible to separate and purify the enzymes while being immobilized.
3. The enzymes are not usually deactivated by adsorption.
4. The adsorption is a reversible process. Immobilized Enzyme 53
However, adsorption techniques also have several disadvantages:
1. The bonding strength is weak.
2. The state of immobilization is very sensitive to solution pH, ionic strength,
and temperature.
3. The amount of enzymes loaded on a unit amount of support is usually low.
A) Entrapment:
B) ADSORPTION:
i) Conventional adsorption
ii) Ion exchange
iii) Affinity adsorption
PURIFICATION
TECHNIQUES:
A) PRECIPITATION
B) CHROMATOGRAPHY
C) ELECTROPHORESIS
D) MEMBRANE SEPARARTION :
i) Conventional filtration
ii) By polymeric membranes
iii) Pressure driven membrane separation(see table 10.2)
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