Transcript Immobilization of Enzymes

ERT211 Biochemical Engineering
APPLIED ENZYMES
CATALYSIS
Pn Syazni Zainul Kamal
School of Bioprocess Engineering
Immobilized-enzyme technology
Enzymes :
 Protein molecules which serve to accelerate the
chemical reactions of living cells
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Have great specificity and are not permanently
modified by their participation in reactions.
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Since they are not changed during the reactions,
it is cost-effective to use them more than once.
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However, if the enzymes are in solution with the
reactants and/or products it is difficult to
separate them.
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Therefore, if they can be attached to the reactor
in some way, they can be used again after the
products have been removed.
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The term "immobilized" means unable to move
or stationary.
Immobilize enzyme technology
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Immobilization of an enzyme mean :
it has been confined or localized so that it
can be used continuously
Some of intracellular enzymes are membrane
bound.
Immobilized enzyme provide a model system to
mimic and understand the action of some
membrane-bound intracellular enzyme.
Attachment to solid structure or incorporation in
gels
Advantages of immobilized enzyme
technology
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Multiple or repetitive use of a single batch of
enzymes
Easy separation of enzyme from the product
Immobilized enzymes retain it activity much
longer than those in solution
IE have greater thermal stability
IE easily removed from the reaction making it
easy to recycle
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Product is not contaminated with the
enzyme (especially useful in the food and
pharmaceutical industries)
Allow development of multienzyme reaction
system
Method of enzyme immobilization
Method used influence the properties of
the resulting biocatalyst.
 Method of immobilization :
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a) Chemical methods
b) Physical methods
Covalent bonding
Adsorption
Entrapment
Encapsulation
a) Adsorption
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1st enzyme immobilization method
Attachment of enzymes on the surface of
support particles by weak physical forces (eg.
van der waals or dispersion force)
The enzyme molecules get adhered to the
surface of support particles on account of the
spectacular combination of hydrophobic effects
and the critical formation of several salt-linkages
per enzyme molecule
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Active site of the adsorbed enzyme usually
unaffected. Nearly full actv is retained.
Support materials for enzyme adsorption :
Inorganic materials
alumina, silica, porous glass, ceramics,
clay, bentonite
organic materials
cellulose, starch, activated carbon, amberlite,
sephadex, dowex
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Advantages of adsorption method
-
little or no conformational change of the enzyme
or destruction of its active site
Min reagents used & only minimum of activation
steps are required
Enable enzyme immobilization under mild
condition
Possible high retention of enzyme activity since
no chemical modification in contrast with covalent
bonding method
Simple and cheap
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Disadvantages of adsorption method
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Desorption of the protein (enzyme)
resulting from changes in temp, pH and
ionic strength
Immobilized enzyme tend to leak from
carrier bcoz of weak interaction between
enzyme and carrier
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After few decades, modifications of adsorption
method been done eg. Cross linking & covalent
bonding
Adsorption of enzyme may be stabilized by crosslinking with bi or multifunctional reagents :
- glutaraldehyde
- bisbiazobenzidine
- cyanuric chloride
Enzyme were absorbed onto a carrier (support
materials), then cross-linking the adsorbed protein
(enzyme).
Improved mechanical and substrate accessibility
b) Covalent bonding
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Is the retention of enzymes on support surfaces
by covalent bond formation.
Enzyme molecule bind to support material via
certain functional group :
- amino
- carboxyl
- hydroxyl
- sulfhydryl group
Functional group must not be in the active site
Common trick : flooding enzyme solution
with competitive inhibitor prior to covalent
bonding to block the active site of enzyme
 Functional group on support material
activated by chemical reagent :
- cyanogen bromide
- carbodiimide
- glutaraldehyde
- triazine
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a) Enzyme bind to support material with
hydroxyl group (-OH)
eg. support materials : cellulose, agarose
(sepharose), dextran (sephadex)
 Supports of this type may be activated
specifically for the covalent bonding by
subjecting it to treatment with either
cyanogen bromide or triazine.
 The reaction with the enzyme protein in
each instance involves the –NH2 group of
lysine.
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Using Supports with – OH group that are Activated
by Covalent Bonding with Cyanogen Bromide.
Using Supports with – OH group that are Activated
by Covalent Bonding with Triazine.
b) Enzyme bind to support material with
carboxyl group (-COOH)
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eg. CM-Cellulose
Supports of this type may be activated
specifically for the covalent bonding by
subjecting it to treatment with either azide
derivative or carbodiimide
The reaction involves the participation of
amino (– NH2) moiety present in lysine,
cysteine, serine, tyrosine — are also made
use of in the covalent bonding phenomenon
Using Supports with – COOH group that are
Activated by Covalent Bonding with Carbodiimide.
Using Supports with – COOH group that are
Activated by Covalent Bonding with azide
derivative.
c) Enzyme bind to support material with
amino group (-NH2)
Support containing amino group can be
converted to diazonium chloride by
treating with sodium nitrate (NaNO3) &
HCL (diazotization)
 Enzyme protein link with this derivative
forming an azo linkage involving the
tyrosine residue of enzyme protein
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 Immobilization
of Enzymes using Supports with
Specific —NH2 group Involving Formation of
Diazonium Chloride
 Immobilization
of Enzymes using Supports with
Specific —NH2 group Involving Activation with
Glutaraldehyde.
Advantages of covalent bonding
Stable method
 Prevent leakage of protein (enzyme) into
production stream
 Covalent bonding attachment is not
reversed by pH, ionic strength or substrate.
 Wide range of choices is possible by
selecting carrier materials & binding
method.
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Disadvantages of covalent bonding
Expensive
 Complicated in procedures involved
 Active site may be modified through the
chemical reactions used to create covalent
bonding
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c) Entrapment
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Enzyme molecules are held/entrapped within the
appropriate fibers or gels
Matrix entrapment & membrane entrapment
Matrices used :
Polymeric materials
- Ca-alginate
- agar
- K-carrageenin
- Polyacrylamide
- Collagen
solid matrices
- activated carbon
- porous ceramic
- diatomaceous earth
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The matrix can be a particle, a membrane or a
fiber
Entrapment may or may not necessarily be
accomplished via covalent bonding existing
between the enzyme entities (molecules) and the
carrier matrix.
non covalent bonding in entrapment can be
considered as putting the enzyme in a molecular
cage just like a caged bird.
In a situation when the covalent bonding is
needed, the enzyme molecules required to be
treated with synthetic reagents e.g., acryloyl
chloride, cellulose acetate etc.
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Entrapment method using polymer matrix
(non covalent bonding)
1)Enzyme solution mixed with polymer solution
2)Polymerization occur
(covalent bonding needed)
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the enzymes surface lysine residues may be
derivatized by reaction with acryloyl chloride to
give the acryloyl amides
Acryloyl amides are then co-polymerized and
cross-linked with acrylamide and bisacrylamide
to form a gel containing enzyme
Gel containing enzyme may be used to form a
small bead or a film on a solid support
Membrane entrapment
 Hollow fiber units used to entrap an enzyme
solution between thin, semipermiable
membrane
 Membrane used :
- nylon
- cellulose
- polysulfone
- polyacrylate
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Entrapment method using membrane
eg. Using cellulose acetate fiber
Enzyme and cellulose acetate is blended
together to obtain an ‘emulsion’ in an
organic solvent, methylene chloride
The resulting emulsion is subjected to the
process of ‘extrusion’ to obtain fibres into a
solution of an aqueous precipitant
d) Encapsulation
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Encapsulation or microencapsulation or
membrane confinement is another effective
approach of enzyme immobilization
A special form of membrane entrapment
A droplet of Enzyme are entrapped in small
capsules (diameter up to 300µm)
The capsules surrounded by spherical
membrane
The membrane have pores permitting small
substrate & product molecules to enter and
leave the capsules
However the pores are too small for
enzyme & other large molecules to
penetrate
 2 type of membrane can be made:
a) permanent polymeric membrane
b) nonpermanent microcapsules
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