Enzyme Immobilization

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by amit gup

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Enzyme Immobilization:

Enzyme Immobilization

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It is defined as imprisonment or physical confinement of enzyme or enzyme containing material in a distinct phase separated from bulk phase (that allows exchange with) containing substrate, effector or inhibitor molecules. The reaction can be controlled and monitored

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Advantages of immobilization: The immobilised material can be reused and gives process economy The products can be easily separated Immobilization can confer stability to the enzyme The reaction can be controlled and monitored at will

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Methods of immobilization: Adsorption Covalent bonding Crosslinking Entrapment Encapsulation

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Support binding and Crosslinking

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Entrapment and Encapsulation

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Adsorption: Earliest method of immobilization A range of non-specific or specific bonding forces may be employed Example electrostatic, hydrophobic interactions or affinity bonding to specific ligands attached to polymer Advantages : Ease and mild conditions of preparation. Simply stir the enzyme and polymer together

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Availability of ready prepared polymer material for use in column reactors Potential for regeneration Limitation of access of the bound enzyme to the substrate is negligible Drawbacks: Leaching of enzyme or cells occurs relatively easily with changes in pH or raised ionic strength Substrates carrying same charge as the polymer may be prevented due to ionic partitioning

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2. Covalent bonding: There are two ways of covalently bonding an enzyme to a polymer – first by activating the polymer with a reactive group, the second by use of bifunctional reagent to bridge between enzyme and polymer The groups on enzyme used for coupling include hydroxyl, amino and lesser extent sulfhydryl groups. Major problem – enzyme may be inactivated by conformational change after reaction or by reaction with a group at the active site

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The commonest forms of activated polymer are hydrogels (celluloses or polyacrylamides) which have diazo , carbodiimide or azide groups Hydrogels can also be directly activated with cyanogen bromide Sometimes polymer, enzyme and reagent such as glutaraldehyde can be mixed simultaneously to yield the immobilized material

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The advantage of this method over adsorption is that leaching of the catalyst may not be a problem apart from the other advantages of adsorption The disadvantages include – use of toxic agents, frequent inactivation of the enzyme, complicated preparative routines and inability to yield immobilized cell preparations 3. Crosslinking: Crosslinking of the enzyme itself by reaction with a bifunctional reagent

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Bifunctional reagent is most often glutaraldehyde Has all the potential advantages of covalent bonding with the associated problems Technique is cheap, simple but not often used with pure proteins, as it yields little bulk of immobilized enzyme with very high intrinsic activity Widely used for commercial preparations of glucose isomerase

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4. Entrapment: Entrapment in a polymer matrix in principle is easy to perform The method is by polymerisation reaction in which the matrix forms over the enzyme Calcium alginate immobilization is the method of choice Polymers which have wide application include- polyacrylamide, cellulose triacetate, agar, gelatin, carrageenan and alginate

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Major advantages – simple method, use of mild conditions, useful for preparing whole cell immobilized materials Disadvantages include – diffusional limitation and leakage of the enzyme through the pores of the gel 5. Encapsulation: In this method a droplet of the enzyme solution is enclosed in a semipermeable membrane capsule Method used for medical application

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Method is simple and cheap. However for the method to be effective the biocatalyst should be stable in solution Materials used include nylon or biodegradable polylactic acid or phospholipid liposomes Advantages – a very high surface area to volume ratio, easy to perform, allows easy replacement of the enzyme in bioreactors

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Disadvantages- 1. Enzyme is not protected as in other methods and there is no potential for stabilization of the enzyme Adsorption of the enzyme on the walls of the fiber may be accompanied by denaturation 2. Application of technique limited by the molecular weight of the substrate

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