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Modh
Assistant professor at ssscp ,zundal
DEFINATION
Immobilization is define as the imprisonment of cell or enzyme in a
distinct support or matrix.
1. Static Method: In this method the carrier is just dipped and left in
the enzyme solution without any stirring.
2. Dynamic Method: In this method carrier is placed in the enzyme
solution and stirring is maintained to load the enzyme on carrier
surface.
3. Reactor loading Method: In this method the carrier is placed in
reactor and enzyme solution is loaded with continuous stirring.
4. Electrode position Method: In this method carrier is put in
vicinity of an electrode. Now the enzymes migrate to carrier in
presence of electric current.
Advantages
No pore diffusion limitation.
Easy to carry out
No reagent are required
Minimum activation steps involved
Comparatively cheap method
Less distruptive to enzyme
disadvantages
Desorption of enzymes from the carrier
Efficiency is less
Covalent bondind
This method involves the formation of covalent bonds
between the chemical groups in enzyme and to the
chemical groups on the support or carrier.
Widely used method.
Hydroxyl group and amino groups of support or enzyme
form covalent bonds more easily.
The formation of colvalent bond usually takes place
particularly with the side chains of amino acids present in
the enzyme ; however, their actual strength of reactivity
being exclusively linked to the status of ‘charge present in
them as given below :
— S– > — SH > — O– > — NH2 > — COO– > — OH >> —
NH3
Thus, the various functional moieties mostly present
in enzyme which actively take part in the formation of
the numerous viable chemical bonds are : sulphide,
sulphhydril, oxide, amino, carboxyl,hydroxyl,
ammonium, imino, amide, methylthiol, guanidyl,
imidazole, and phenol ring.
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, whereby 3D-
network may be obtained by cross-linking with low molecular
weight bifunctional agent(s).
In doing so, 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.
Importantly, such an overwhelmingly loss in the enzymatic
activity may be overcome by judiciously carrying out the
‘enzyme immobilization’ either in the presence of a
competitive inhibitor or an enzyme substrate.
Carriers or support commonly
used for covalent bond
Carbohydrates:- eg.cellulose, DEAD cellulose, agarose
Synthetic agents:-eg. Polyacrylamide
Protein carriers:- collagen, gelatin
Amino group bearing carriers:- eg.amino benzyl
cellulose
Inorganic carriers:porous glass,silica
Cyanogen bromide: eg.agarose and CN Br sepharose
Methods of covalent bonding
1. Diazoation:- bonding between amino group of
support and tyrosil or histidine group of enzyme.
2. Peptide bond: bonding between amino or carboxyl
group of the support and that of the enzyme.
3. Poly functional reagents:use of bi-functional or
multifunctional reagent (glutaraldehyde) which
forms covalent bonds between the amino group of
the support and amino group of the enzyme.
advantages
Adsorption of enzymes to the carrier matrices is quite
easy and convenient, and hence used extensively.
Covalent bonding attachment is not reversed by pH,
ionic strength or substrate.
Relatively broader spectrum of bonding reactions, and
of matrices with functional moieties capable of either
having covalent bondage or prone to be activated to
yield such groups renders this method into a highly
acceptable one.
disadvantages
Chemical modifiaction of enzymeleading to the lose of
functional conformation of enzyme.
Enzyme inactivation by change in the conformation
when undergoes reactions at the active site.
Entrapment