imobilized enzyme bt

7/31/2019 Imobilized Enzyme Bt

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IMMOBILISEDIMMOBILISED

ENZYMESENZYMES


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ARGUMENT FORARGUMENT FORIMMOBILISATIONIMMOBILISATION

Enzymes are expensive, they should be utilized in anEnzymes are expensive, they should be utilized in anefficient mannerefficient manner

As catalytic molecules, enzymes are not directly usedAs catalytic molecules, enzymes are not directly usedup. After the reaction the enzymes cannot beup. After the reaction the enzymes cannot be

economically recovered for re-use and are generallyeconomically recovered for re-use and are generallywastedwasted This enzyme residue remains to contaminate theThis enzyme residue remains to contaminate the

product and its removal may involve extra purificationproduct and its removal may involve extra purificationcostscosts

Simple and economic methods must be used

to separate the enzyme from the reaction product


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Immobilization of enzymes refers to the technique ofImmobilization of enzymes refers to the technique ofconfining/anchoring the enzymes in or on inert supportconfining/anchoring the enzymes in or on inert supportfor their stability and functional reusefor their stability and functional reuse

Advantages of immobilized enzymesAdvantages of immobilized enzymes Stable and efficient in functionStable and efficient in function Can be reused again and againCan be reused again and again Products are enzyme freeProducts are enzyme free Ideal for multiple enzyme reaction systemsIdeal for multiple enzyme reaction systems Control of enzyme function is easyControl of enzyme function is easy Suitable for industrial and medical useSuitable for industrial and medical use Minimize effluent disposal problemsMinimize effluent disposal problems

Disadvantages:Disadvantages: Loss of biological activityLoss of biological activity expensiveexpensive


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METHODS OF IMMOBILISATIONMETHODS OF IMMOBILISATION

1.1. AdsorptionAdsorption

2.2.

Covalent bindingCovalent binding

3.3. EntrapmentEntrapment

4.4. Membrane confinementMembrane confinement


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METHODS OF IMMOBILISATIONMETHODS OF IMMOBILISATION


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MATRICES FOR ENZYME IMMOBILISATIONMATRICES FOR ENZYME IMMOBILISATION

1.1. Inert.Inert.2.2. Physically strong and stable.Physically strong and stable.

3.3. Should be cheap enough to discard.Should be cheap enough to discard.

4.4. Better if it could be regeneratedBetter if it could be regeneratedafter the useful lifetime of theafter the useful lifetime of theimmobilised enzyme.immobilised enzyme.

5.5. The surface available to the enzyme.The surface available to the enzyme.


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ADSORPTION OF ENZYMESADSORPTION OF ENZYMESONTO INSOLUBLE SUPPORTSONTO INSOLUBLE SUPPORTS

Physical binding of enzymes on the surface of anPhysical binding of enzymes on the surface of aninert supportinert support

Support materials- inorganic (alumina, silica gel,Support materials- inorganic (alumina, silica gel,

calcium phosphate gel, glass)calcium phosphate gel, glass) Organic starch, carboxymethyl cellulose, DEAE-Organic starch, carboxymethyl cellulose, DEAE-

cellulose, DEAE- sephadexcellulose, DEAE- sephadex Weak forces - vander waals force and hydrogenWeak forces - vander waals force and hydrogen

bondsbondsAdsorbed enzymes- easily removed by changingAdsorbed enzymes- easily removed by changing

pH,ionic strength or temperaturepH,ionic strength or temperature


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EXAMPLES OF SUITABLEEXAMPLES OF SUITABLEADSORBENTSADSORBENTS

Ion-exchange matricesIon-exchange matrices Porous carbonPorous carbon

ClaysClays Hydrous metal oxidesHydrous metal oxides GlassesGlasses Polymeric aromatic resinsPolymeric aromatic resins


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ENTRAPMENT OF ENZYMESENTRAPMENT OF ENZYMESWITHIN GELS OR FIBRESWITHIN GELS OR FIBRES

Purely physical caging: size of the matrix pore isPurely physical caging: size of the matrix pore issuch that the enzyme is retained, substrate andsuch that the enzyme is retained, substrate andpdt pass thrupdt pass thru

Tech commonly referred lattice entrapmentTech commonly referred lattice entrapmentMatrices used- polyacrylamide gel, collagen,Matrices used- polyacrylamide gel, collagen,gelatin, starchgelatin, starch

Entraped cells used- production of aminoacids (L-Entraped cells used- production of aminoacids (L-isoleucine,L-aspartic acid), L-malic acid andisoleucine,L-aspartic acid), L-malic acid andhydroquinonehydroquinone


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MicroencapsulationMicroencapsulation

It is a type of entrapment refers to theIt is a type of entrapment refers to the

process of spherical particle formationprocess of spherical particle formation

wherein a liquid or suspension is enclosedwherein a liquid or suspension is enclosed

in a semipermeable membranein a semipermeable membrane

Membrane-polymeric,lipoidal,lipoproteinMembrane-polymeric,lipoidal,lipoprotein

based or non-ionic in naturebased or non-ionic in nature


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Three distinct ways of microencapsulationThree distinct ways of microencapsulation

Building of special membrane reactorsBuilding of special membrane reactors Formation of emulsionsFormation of emulsions

Stabilization of emulsions to formStabilization of emulsions to form

microcapsulesmicrocapsules

It is recently used for immobilization ofIt is recently used for immobilization of

enzymes and mammalian cellsenzymes and mammalian cells


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Covalent bindingCovalent binding

Covalent bond b/w chemical groups ofCovalent bond b/w chemical groups of

enzymes and the chemical groups ofenzymes and the chemical groups of

supportsupport

It is often associated with loss of enzymeIt is often associated with loss of enzyme

activityactivity

Inert support needs pretreatment before itInert support needs pretreatment before it

binds enzymesbinds enzymes


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Common methods of covalentCommon methods of covalent

bindingbinding

Cyanogen bromide activation:Cyanogen bromide activation:

Inert support materials-Inert support materials-

cellulose,sepharose,sephadex containingcellulose,sepharose,sephadex containing

glycol groups activated by CNBr,whichglycol groups activated by CNBr,which

then binds to enzymes and immobilizethen binds to enzymes and immobilize

themthem


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DiazotationDiazotation

Support materials-aminobenzyl cellulose,Support materials-aminobenzyl cellulose,

amino derivatives of polystyrene,amino derivatives of polystyrene,

aminosilanized porous glassaminosilanized porous glass

These are subjected to diazotation onThese are subjected to diazotation on

treatment with NaNO2 and HCltreatment with NaNO2 and HCl

In turn bind covalently to tyrosyl orIn turn bind covalently to tyrosyl or

histidyl groups of enzymeshistidyl groups of enzymes


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Peptide bond formationPeptide bond formation

Peptide bond b/w amino or carboxylPeptide bond b/w amino or carboxyl

groups of the support and carboxy orgroups of the support and carboxy or

amino group of enzymeamino group of enzyme

Support material is chemically treated toSupport material is chemically treated to

form active functional groupsform active functional groups


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Activation by bi- or polyfunctionalActivation by bi- or polyfunctional

reagentsreagents

Some of the reagents such asSome of the reagents such as

glutaraldehyde used to create bonds b/wglutaraldehyde used to create bonds b/w

amino groups of enzymes and aminoamino groups of enzymes and amino

groups of supportgroups of support Eg., aminoethylcellulose,Eg., aminoethylcellulose,

albumin,aminoalkylated porous glassalbumin,aminoalkylated porous glass


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Cross linkingCross linking

The absence of solid supportThe absence of solid support

Enzymes immobilized by creating cross-linksEnzymes immobilized by creating cross-links

between them,thru polyfunctional reagentsbetween them,thru polyfunctional reagents

Reagents react with enzymes and create bridgesReagents react with enzymes and create bridgeswhich form the backbone to hold enzymeswhich form the backbone to hold enzymes

Crosslinking reagents: glutaraldehyde,Crosslinking reagents: glutaraldehyde,

diazobenzidine, hexamethylene diisocyanate anddiazobenzidine, hexamethylene diisocyanate andtoluene diisothiocyanatetoluene diisothiocyanate


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Choice of immobilization techniqueChoice of immobilization technique

Trial and error methodTrial and error method

Factors to decide a techniqueFactors to decide a technique

Catalytic activityCatalytic activity StabilityStability

RegenerabilityRegenerability

Cost factorCost factor


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The most commonly used method forThe most commonly used method for

immobilizing enzymes on theimmobilizing enzymes on theresearch scale involves Sepharoseresearch scale involves Sepharose(poly-{(poly-{-1,3-D-galactose--1,3-D-galactose--1,4-(3,6--1,4-(3,6-

anhydro)-L-galactose}), activated byanhydro)-L-galactose}), activated byCyanogen bromide, because it is aCyanogen bromide, because it is acommercially available beadedcommercially available beaded

polymer which is highly hydrophilicpolymer which is highly hydrophilicand generally inert to microbiologicaland generally inert to microbiologicalattackattack


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IMMOBILIZED ENZYME ACTIVITYIMMOBILIZED ENZYME ACTIVITYDEPENDS ON CORRECT ANDDEPENDS ON CORRECT AND

UNSTRAINED CONFORMATIONUNSTRAINED CONFORMATION


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CONFINEMENT OF ENZYMESCONFINEMENT OF ENZYMESINSIDE A MEMBRANEINSIDE A MEMBRANE

Semipermeable membrane: Hollow fibreSemipermeable membrane: Hollow fibremembranemembrane

Membrane-bound droplets: Nylon-6,6Membrane-bound droplets: Nylon-6,6Liposome: PhospholipidLiposome: Phospholipid


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GENERALISED COMPARISON OF DIFFERENTGENERALISED COMPARISON OF DIFFERENTENZYME IMMOBILISATION TECHNIQUESENZYME IMMOBILISATION TECHNIQUES

Characteristics Adsorption Covalent

binding

Entrapment Membrane

confinement

Preparation Simple Difficult Difficult Simple

Cost Low High Moderate HighBinding force Variable Strong Weak Strong

Enzyme leakage Yes No Yes No

Applicability Wide Selective Wide Very wide

Running Problems High Low High High

Matrix effects Yes Yes Yes No

Large diffusional

barriers

No No Yes Yes

Microbial protection No No Yes Yes


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Enzyme EC number Product

Aminoacylase 3.5.1.14 L-Amino acids

Aspartate ammonia-lyase 4.3.1.1 L-Aspartic acid

Aspartate 4-decarboxylase 4.1.1.12 L-Alanine

Cyanidase 3.5.5.x Formic acid (from waste cyanide)

Glucoamylase 3.2.1.3 D-Glucose

Glucose isomerase 5.3.1.5 High -fructose corn syrup

Histidine ammonia-lyase 4.3.1.3 Urocanic acid

Hydantoinasea 3.5.2.2 D- and L-amino acids

Invertase 3.2.1.26 Invert sugar

Lactase 3.2.1.23 Lactose-free milk and whey

Lipase 3.1.1.3 Cocoa butter substitutes

Nitrile hydratase 4.2.I.x Acrylamide

Penicillin amidases 3.5.1.11 Penicillins

Raffinase 3.2.1.22 Raffinose-free solutions

Thermolysin 3.2.24.4 Aspartame

Some of themore

important

industrialuses of

immobilised

enzymes


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Immobilization of L-aminoacidImmobilization of L-aminoacid

acylaseacylase

First enzyme to be immobilizedFirst enzyme to be immobilized

40 different methods attempted40 different methods attempted

3 methods found useful3 methods found useful Covalent binding to iodoacetyl celluloseCovalent binding to iodoacetyl cellulose

Ionic binding to iodoacetyl celluloseIonic binding to iodoacetyl cellulose

Ionic binding to DEAE-sephadex andIonic binding to DEAE-sephadex andentrapment within polyacrylamideentrapment within polyacrylamide


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Stabilization of soluble enzymesStabilization of soluble enzymes

Some enzymes- cannot be immobilizedSome enzymes- cannot be immobilized

and have to be used in soluble formand have to be used in soluble form

Such enzymes-stabilized by usingSuch enzymes-stabilized by using

additives or by chemical modificationadditives or by chemical modification

Stabilized enzymes - longer half - lives,Stabilized enzymes - longer half - lives,


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Methods of enzyme stabilizationMethods of enzyme stabilization

Solvent stabilization: solvents at low concSolvent stabilization: solvents at low conc

stabilize enzymesstabilize enzymes

Substrate stabilization: active site can beSubstrate stabilization: active site can be

stabilized by adding substratesstabilized by adding substrates

Stabilization by polymers: enzymesStabilization by polymers: enzymes

stabilized against increased temperaturestabilized against increased temperature

by addition of polymers such as gelatin,by addition of polymers such as gelatin,

albumin and polyethylene glycolalbumin and polyethylene glycol


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Stabilization by saltstabilization by salts :: Stability of metalloenzymes achieved by adding saltsStability of metalloenzymes achieved by adding salts

Ca,Fe,Mn,Cu and ZnCa,Fe,Mn,Cu and Zn Eg protease can stabilized by adding caEg protease can stabilized by adding caStabilization by chemical modificationtabilization by chemical modification :: Addition of polyamino side chain Eg., poly tyrosine, polyAddition of polyamino side chain Eg., poly tyrosine, poly

glycineglycine

Acylation of enzymes by adding groups such as acetyl,Acylation of enzymes by adding groups such as acetyl,propoinyl and succinylpropoinyl and succinyl

Stabilization by rebuilding:tabilization by rebuilding: Theoretically the stability of the enzymes due toTheoretically the stability of the enzymes due to

hydrophobic interactions in the core of the enzyme. It ishydrophobic interactions in the core of the enzyme. It is

therefore proposed by enhancing hydrophobictherefore proposed by enhancing hydrophobicinteractions it can be stabilizedinteractions it can be stabilized

the enzymes is first unfold and rebuiltthe enzymes is first unfold and rebuilt


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The enzymes is first unfold and rebuiltThe enzymes is first unfold and rebuilt

Enzyme is first chemically treated andEnzyme is first chemically treated and

then refoldedthen refolded

Refolding can be done in presence of lowRefolding can be done in presence of low

mol wgt ligandsmol wgt ligands

For certain enzymes, refolding at higherFor certain enzymes, refolding at highertemp stabilizes themtemp stabilizes them


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Stabilization by site-directedStabilization by site-directed

mutagenesismutagenesis

site-directed mutagenesis used to producesite-directed mutagenesis used to produce

more stable and functionally moremore stable and functionally moreefficient enzymesefficient enzymes


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Immobilization of cellsImmobilization of cells

Immobilized individual enzymes-used forImmobilized individual enzymes-used forsingle step reactionsingle step reaction

They are not suitable for multienzymeThey are not suitable for multienzyme

rxns requiring cofactorsrxns requiring cofactors Whole cells or cellular organelles-Whole cells or cellular organelles-

immobilized to serve as multienzymeimmobilized to serve as multienzyme

systemssystems Sometimes cells are used for single stepSometimes cells are used for single step

rxn due to cost factor in isolating enzymesrxn due to cost factor in isolating enzymes


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Immobilized viable cellsImmobilized viable cells

Viability of the cells preserved by mildViability of the cells preserved by mild

immobilizationimmobilization

Immobilized cells used for fermentationImmobilized cells used for fermentation

Mammalian cells- used as immobilizedMammalian cells- used as immobilized

viable cellsviable cells


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Immobilization non-viable cellsImmobilization non-viable cells

It is prefered over the enzymes or evenIt is prefered over the enzymes or even

the viable cellsthe viable cells

Becoz of costly isolation and purificationBecoz of costly isolation and purification

processesprocesses

Eg., immobilization of cells containingEg., immobilization of cells containing

glucose isomerase for the industrialglucose isomerase for the industrial

production of fructose syrupproduction of fructose syrup


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Limitations of immobilizingLimitations of immobilizing

eukaryotic cellseukaryotic cells

Due to the presence of cellular organellesDue to the presence of cellular organelles

the metabolism is slowthe metabolism is slow

It is used for the production of complexIt is used for the production of complex

proteins such as immunoglobulinsproteins such as immunoglobulins

For the proteins that undergo postFor the proteins that undergo post

translational modificationtranslational modification

Usually prokaryotes fit well for industrialUsually prokaryotes fit well for industrial

production of biochemicalsproduction of biochemicals


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Effect of immobilization on enzymeEffect of immobilization on enzyme

propertiesproperties

Enzyme immobilization associated withEnzyme immobilization associated with

alterations in enzyme propertiesalterations in enzyme properties

Substantial decrease in enzyme specificitySubstantial decrease in enzyme specificity

due to conformational change occurdue to conformational change occur

during immobilizationduring immobilization

Kinetic constants Km and Vmax of anKinetic constants Km and Vmax of an

native enzyme different from that of thenative enzyme different from that of the

immobilzed enzymeimmobilzed enzyme


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Immobilized enzyme reactorsImmobilized enzyme reactors

Immobilized enzyme cells are utilized inImmobilized enzyme cells are utilized in

the industrial processes in the form ofthe industrial processes in the form of

enzyme reactorsenzyme reactors

Batch reactorsBatch reactors

Continuous reactorsContinuous reactors

Membrane reactorsMembrane reactors


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Batch reactorsBatch reactors

Immobilized enzymes and substrates placedImmobilized enzymes and substrates placed

Reaction is allowed to takes place underReaction is allowed to takes place under

constant stirringconstant stirring

As rxn completes the pdt is separated fromAs rxn completes the pdt is separated fromenzymeenzyme

Soluble enzymes commonly used in batchSoluble enzymes commonly used in batch

fermentationfermentation Difficult to separate enzyme and limitation ofDifficult to separate enzyme and limitation of

their reusetheir reuse


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Stirred tank reactorsStirred tank reactors

Composed of reactor with stirrer- allowsComposed of reactor with stirrer- allows

good mixing and appropriate temperaturegood mixing and appropriate temperature

and pHand pH

Chance of loss of enzyme activityChance of loss of enzyme activity

Modification of stirrer tank is basketModification of stirrer tank is basket

reactorreactor

Enzyme is retained in the impeller bladesEnzyme is retained in the impeller blades


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Plug flow type reactorsPlug flow type reactors

The flow rate of fluids controlled by a plugThe flow rate of fluids controlled by a plug

systemsystem

It is in the form of packed bed or fluidizedIt is in the form of packed bed or fluidized

bedbed

Useful for obtaining kinetic data on theUseful for obtaining kinetic data on the

reaction systemsreaction systems


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Continuous reactorsContinuous reactors

Substrate added continuously and productSubstrate added continuously and product

removed simultaneouslyremoved simultaneously

AdvantagesAdvantages

Control over product formationControl over product formation

Convenient operation of the systemConvenient operation of the system

Easy automation of the entire processEasy automation of the entire process


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Membrane reactorsMembrane reactors

Membrane materials include polysulfone,Membrane materials include polysulfone,polyamide and cellulose acetatepolyamide and cellulose acetate

Biocatalyst normally retained on theBiocatalyst normally retained on the

membranes of the reactormembranes of the reactor Recycle model membrane reactor, theRecycle model membrane reactor, the

contents, cofactors and substrates alongcontents, cofactors and substrates along

with the freshly released product- recycledwith the freshly released product- recycledusing a pumpusing a pump The product passes out can be recoveredThe product passes out can be recovered


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Applications of immobilised enymesApplications of immobilised enymes

and cellsand cells

Manufacturing of commercial productsManufacturing of commercial products Production of L-aminoacids are important for useProduction of L-aminoacids are important for use

in food, feed and medical purposein food, feed and medical purpose

Chemical methods employed for their productionChemical methods employed for their productionresult in racemic mixture of D and L-aminoacidsresult in racemic mixture of D and L-aminoacids They are then acylated to form D,L-acylThey are then acylated to form D,L-acyl

aminoacidsaminoacids

Immobilised enzyme aminoacylase selectivelyImmobilised enzyme aminoacylase selectivelyhydrolyse D,L-acyl aminoacids to form L-hydrolyse D,L-acyl aminoacids to form L-aminoacidsaminoacids


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Production of high fructose syrupProduction of high fructose syrup

High fructose syrup contains approxHigh fructose syrup contains approx

equivalent amount of glucose and fructoseequivalent amount of glucose and fructose

HFS is a good substitute for sugarHFS is a good substitute for sugar

HFS can be produced from glucose byHFS can be produced from glucose by

employing an immobilized enzyme glucoseemploying an immobilized enzyme glucose

isomeraseisomerase


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Immobilized enzymes and cellsImmobilized enzymes and cells

analytical applicationsanalytical applications

Enzyme electrode- glucometerEnzyme electrode- glucometer

Substance such as urea, cholesterol,Substance such as urea, cholesterol,

lactate, alcohol can be assayedlactate, alcohol can be assayed

In affinity chromatography immobilizedIn affinity chromatography immobilized

enzymes- used to purify severalenzymes- used to purify several

compounds, antigens and antibodiescompounds, antigens and antibodies

imobilized enzyme bt

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