Download Immobilization of Enzymes

Dr. A.K.M. Shafiqul Islam
School of Bioprocess Engineering
19.01.10
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Why we need Immobilization?
In actual practice, the soluble enzymes
engaged in ‘batch operations’ is found to be
not-so-economical due to the fact that the
active enzyme is virtually lost (not recovered)
after each viable reaction.
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Therefore, in order to combat and overcome
such a non-productive, economically not
feasible, and deleterious effect the enzymes
have been ultimately immobilized ;
and the process is termed as enzyme
immobilization.
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Immobilization is defined as — ‘confining the
enzyme molecules to a distinct phase from
the one wherein the substrates and the
products are present’.
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Immobilization of an enzyme means that it
has been confined or localized so that it can
be reused continuously
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Enzymes are more or less physically confined in the
course of a definite continuous catalytic process.
They may be suitably recovered from the reaction
mixture and used over and over again thereby
gainfully improving the economic viability of the
entire process.
It may be accomplished by fixing the enzyme
molecules to or within certain appropriate
subtance.
It should be absolutely critical that both the
substrates and the products migrate quite freely in
and out of the phase to which the specific enzyme
molecules are actually confined.
Certain enzymes which are as such readily
inactivated by heat (i.e., thermolabile), may be
rendered heat-stable by attachment to inert
polymeric supports.
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Immobilized enzymes may be recycled, rapidly
controlled, operated continuously, product(s)
easily separable, and above all the enzymatic
properties (i.e., stability, activity) altered
favourably.
Immobilized enzymes seem to be fairly
compatible with multienzyme procedures that
essentially make use of enzyme-based
cofactors.
Utilization of whole beads are invariably
employed for a wide spectrum of catalytic
functions thereby ascertaining the fact that
immobilized enzymes do have an edge over
their soluble counterparts overwhelmingly.
1. For processing with isolated enzymes,
 an immobilized form can be retained in the reactor
2. In solution, some enzymes will leave the reactor
with the final product.
 New enzymes be introduced to replace the lost ones,
3. Enzymes in the product may be undesirable
impurities which must be removed.
4. Immobilized enzymes may retain their activity
longer than those in solution
5. An immobilized enzyme may be fixed in position
near other enzymes participating in a catalytic
sequence,
 thereby increase the catalyst efficiency for the
multistep conversion.
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Advantages of immobilization
◦ Enzyme reutilization
◦ Elimination of enzyme recovery and purification
◦ May provide better environment for activity
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Methods of immobilization
◦ Surface immobilization
 Adsorption
 covalent binding
 Ionic bonding
◦ Entrapment
◦ Encapsulation
Where weaker interactions or containment of the
enzymes are involved.
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Enzymes may be adsorbed on a variety of carriers
offering in some cases the practical convenience
of simple regeneration by removal of deactivated
enzyme and reloading with fresh, active catalyst.
If a support or entrapping material is used, its
properties combined with those of the enzyme
and the immobilization procedure dictate overall
catalyst properties.
Where covalent bonds are formed with the enzyme
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If the native surface is not ideal, can it be chemically
modified or coated to facilitate enzyme attachment.
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These materials have been employed for covalent
enzyme immobilization
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They have some interesting surface functional
groups.
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materials which have been used as immobilized
enzyme supports include ceramics, glass and other
metal oxides.
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chemical methods
◦ where covalent bonds
are formed with the
enzyme
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physical methods
◦ where weaker
interactions or
containment of the
enzymes are involved
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Four types of immobilization
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Adsorption Method
Covalent bonding
Entrapment
Encapsulation
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By allowing the contact of the enzyme and
the polymer support either by percolating the
enzyme via a packed bed, tube, membrane
formed from a support material or in a stirred
bioreactor.
Percolating - Filtering especially through
small holes or crevices.
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The enzyme molecules get adhered to the
surface of a carrier matrix on account of the
spectacular combination of hydrophobic
effects and the critical formation of several
salt-linkages per enzyme molecule
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The covalent bonding of an enzyme may be
accomplished either by activating the polymer
with a reactive moiety (i.e., copolymerization
with ethylene, anhydride of maleic acid)
or by effectively employing the bifunctional
reagent to serve as a bridge between the two
entities : enzyme and polymer
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In such case, the enzyme invariably may get
inactivated because the reactions normally
engage a functional moiety strategically
located at the ‘active site’ of the enzyme.
Thus, the overall net effect being the
substantial loss of enzymatic activity.
The loss in enzymatic activity may be
overcome by carrying out the ‘enzyme
immobilization’ either in the presence of a
competitive inhibitor or an enzyme substrate.
Natural supports
Synthetic supports
Cellulose (—OH)
CM-cellulose (—COON)
Agarose (Sepharose) (—OH)
Polyacrylamide derivatives
(Bio-Gel, Enzacryl) (-aromatic amino)
Polyaminopolystyrene (—NH2)
Dextran (Sephadex) (—OH)
Malefic anhydride copolymers
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Adsorption of enzymes to the carrier matrices
is quite easy and convenient, and hence used
extensively.
Covelent bonding attachment is not reversed
by pH, ionic strength or substrate.
Relatively broader spectrum of bonding
reactions, and of matrices with functional
group capable of either having covalent
bondage or prone to be activated to yield
such groups renders this method into a
highly acceptable one.
Interaction
Adsorbents
Physical
adsorption
Activated carbon, silica gel, alumina, starch, clay,
glass
Modified materials
tannin-ammohexyl cellulose, Concanavalin A-
Ionic binding
Sepharose
Cation exchangers
CM-cellulose, Aberlite, CG-50, Dowex 50
Anion exchangers
DEAF-cellulose, DEAF-Sephadex,
polyarnmopolystyrene, Amberlite IR-45
CM = carboxymethyl; DEAF = diethylaminoethyl.
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Enzyme in solution can be used once
It can be fixed on a carrier so can be used
continuously
It can be bound, adsorbed, entrapped or
crosslinked
They are more heat stable, pH is shifted