immobilised enzymes.pdf enzyme technology

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1. Immobilised enzymes
•When purified enzymes are used to make large quantites
of another product, downstreamprocessing can be
difficult and expensive.
•Immobilising enzymes is cheaper.
•Enzyme molecules are attached to a support matrix rather
than free in solution. They still function properly but can
be kept separate from the reactants and the products.
•Immobilisedenzymes are usually used in continuous flow-
through re-actors, which have a low volume
.
•Isolated enzymes can be immobilised so that they do
not contaminate the end product and can be used
again and again

2. Methods of Enzyme Immobilization
There are five different techniques of immobilizing enzymes :
(i)Adsorption-Anenzymemaybeimmobilizedbybondingtoeither
externalorinternalsurfaceofacarrierorsupportsuchasmineral
support(aluminiumoxide,clay),organicsupport(starch),modified
sapharoseandionexchangeresins.Bondsoflowenergyareinvolved
e.g.ionicinteractions,hydrogenbonds,vanderWaalsforces,etc.
(ii)Covalentbonding-Covalentbondisformedbetweenthechemical
groupsofenzymeandchemicalgroupsonsurfaceofcarrier.Covalent
bondingisthusutilizedunderabroadrangeofpH,ionicstrengthand
othervariableconditions.Immobilizationstepsareattachmentof
couplingagentfollowedbyanactivationprocess,orattachmentofa
functionalgroupandfinallyattachmentoftheenzyme

3. useofcyanogenbromidetoasupportcontainingglycolgroupi.e.cellulose,
syphadex,sepharose,etc.Useofabifunctionalormultifunctionalreagent
e.g.glutaraldehydewhichformsbondingbetweentheaminogroupofthe
supportandaminogroupoftheenzyme
(iii)Entrapment-Enzymescanbephysicallyentrappedinsideamatrix(support)
ofawatersolublepolymersuchaspolyacrylamidetypegelsandnaturally
derivedgelse.g.cellulosetriacetate,agar,gelatin,carrageenan,alginate,etc
i)inclusioningels(enzymeentrappedingels),(ii)inclusioninfibers
(enzymeentrappedinfiberformat),and(iii)inclusioninmicrocapsules
(enzymesentrappedinmicrocapsulesformedmonomermixturessuchas
polyamineandpolybasicchloride,polyphenolandpolyisocyanate).
(iv)Copolymerizationorcross-linking-Cross-linkingischaracterizedbycovalent
bondingbetweenthevariousmoleculesofanenzymeviaapolyfunctional
reagentsuchasglutaraldehyde,diazoniumsalt,hexamethylenedisocyanate,
andN-N'ethylenebismaleimide.

4. (v)encapsulation-Encapsulationistheenclosingofadropletofsolution-of
enzymeinasemipermeablemembranecapsule.Thecapsuleismadeupof
cellulosenitrateandnylon.Themethodofencapsulationischeapandsimple
butitseffectivenesslargelydependsonthestabilityofenzymealthoughthe
catalystisveryeffectivelyretainedwithinthecapsule.Thistechniqueis
restrictedtomedicalsciencesonly

5. Direct ELISA: Antigen is attached to a polystyrene plate. Enzyme-labeled antibody is
added that can react with the antigen and a substrate that can be measured.
Indirect ELISA: Antigen is attached to a polystyrene plate. Addition of primary antibody
followed by an enzyme-labeled antibody that can react with both the primary antibody
and substrate.
Sandwich ELISA: A capture antibody is attached to the polystyrene plate, then antigen is
added that specifically attaches or captures the antigen. A second antibody, also specific
for the antigen but not the same as the capture antibody is added and “sandwiches” the
antigen. This second antibody is then followed by an enzyme-labeled antibody specific
for the second antibody that can react with a substrate that can be measured.
Competitive ELISA: This test is like the sandwich ELISA but involves the addition of
competing antibodies or proteins when the second antibody is added. This results in a
decrease in the substrate signal that is generated. This test is considered to give good,
highly specific results.