Zeta potential measurement as a diagnostic tool in enzyme immobilisation

ColloidsandSurfacesB:Biointerfaces66(2008)39–44

ContentslistsavailableatScienceDirect

ColloidsandSurfacesB:

a

BiosystemsDepartment,Ris?NationalLaboratoryforSustainableEnergy,TechnicalUniversityofDenmark-DTU,Building330,P.O.Box49,DK-4000Roskilde,DenmarkEngler-Bunte-Institute,ChairofWaterChemistry,UniversityofKarlsruhe(TH),Engler-Bunte-Ring1,D-76131Karlsruhe,Germanyc

ResearchCenterKarlsruhe,InstituteofTechnicalChemistry,Hermann-von-Helmholz-Platz1,D-76344Eggenstein-Leopoldshafen,Germanyd

InstituteofEngineeringinLifeScience,TechnicalBiologyUnit,UniversityofKarlsruhe(TH),Engler-Bunte-Ring1,D-76131Karlsruhe,Germany

b

articleinfoabstract

Theef?ciencyofbindingduringenzymeimmobilisationdoesnotonlydependonthechemicalpropertiesoftheenzymeandthematrixparticle,butalsoontheirsurfacepotential.Zetapotentialquanti?estheelectrostaticinteractionsbetweenenzymeandmatrixparticles,andcantherefore,beusedasanindicatorofthebindingef?ciencyintheenzymeimmobilisationstudies.Inordertoestablishacorrelationbetweenthezetapotentialandthebindingef?ciency,weusedCALA(CandidaantarcticaA-typelipase)asamodelproteinforimmobilisationonnon-porousmagneticmicroparticleswithepoxy(M-PVAE02),carboxy(M-PVAC12)andamine(M-PVAN12)terminations.WeobservedmaximalbindingofCALAontotheM-PVAN12beads,duetotheelectrostaticattractionbetweennegativelychargedproteinandcarrierparticleswithslightlypositivezetapotential.ThebindingofCALAwaslowerwhenM-PVAE02beadswereused,followedbyM-PVAC12beads.Thedecreasingbindingef?ciencywasobviouslytheresultofincreasingelectrostaticrepulsionbetweentheinteractionpartners.Thiscouldbecorrelatedtotheincreasingneg-ativezetapotentialofthemagneticparticles.Moreover,themediumofsuspensionoftheparticlesalsomakesasigni?cantdifference.Wefoundhighestspeci?cactivityofthelipaseimmobilisedonM-PVAE02beadsinamediumconcentratedbuffer(0.3M).Theresultsdemonstrateaclearcorrelationbetweenzetapotentialandbindingef?ciencybutnocorrelationbetweenthebeadrelatedspeci?cactivityandthezetapotential.These?ndingsareadvocatingthepossibilityofusingthezetapotentialasadiagnostictoolinenzymeimmobilisation.

?2008ElsevierB.V.Allrightsreserved.

Articlehistory:

Received15January2008

Receivedinrevisedform4April2008Accepted14May2008

Availableonline18May2008Keywords:

Candidaantarcticalipasetype-AEnzyme

ImmobilisationZetapotential

Magneticparticles

1.Introduction

Enzymeimmobilisationhasseveraladvantagessuchaseasyseparationoftheproduct,anditsrecovery,repeatedusabilityoftheenzyme,reducedcosts,etc.Thereforecontinuousprocesseswithimmobilisedenzymesbecomeeconomicallyworthy.Enzymesaretypicallyimmobilisedonlargeporousparticleswhichareeitherpackedincolumnsoraddedbatchwisetoareactionmixtureandarerecoveredbycentrifugationforfurtheruse.Thisprocesshaspotentialproblemssuchasdiffusionlimitationsofthesubstrate[1],dif?cultyofseparationoftheparticlesfromviscousreactionsolutions.Moreover,porousparticlesalsocouldbeeasily“plugged”withreactioncomponents,particularlyincomplexindustrialmix-tures.Alloftheseproblemscanpotentiallybeovercomebyusingnon-porousmagneticallysusceptibleimmobilisedenzymescom-binedwithahigh-gradientmagnetic?shing(HGMF)typeapproachmodi?edforenzymecapture,regenerationandreuse[2,3].Infur-

?Correspondingauthor.Tel.:+497216087059;fax:+497216087051.E-mailaddress:george.metreveli@ebi-wasser.uka.de(G.Metreveli).0927-7765/$–seefrontmatter?2008ElsevierB.V.Allrightsreserved.doi:10.1016/j.colsurfb.2008.05.004

therstudiesahigh-gradientmagneticseparation(HGMS)systemhasbeenadaptedforamini-pilotscalesemi-continuousmulticy-clereuseofCandidaantarcticaA-typelipase(CALA)immobilisedonsuperparamagneticmicroparticles[4].Suchsystemsarebasedonsuperparamagneticparticlesthatwillrespondtomagneticforcesbutwillnotremainpermanentlyaggregatedafterremovaloftheexternal?eld[5].Thesecharacteristics,combinedwiththeeasymagnetisation,makethesuperparamagneticparticlesanidealcorematerialforsupportsdesignedforseparationofbiologicalmateri-alsintendedforrepeateduse.

Currently,thenon-porousparticlesarecommonlyamino-activated,epoxy-activatedorcarboxy-activated.Thecovalentnatureofactivationbondsmakestheseparticlesverystableforenzymeimmobilisation.Theaf?nityofanenzymetoaspeci?ckindofactivatednon-porousparticledoesnotonlydependonthenatureofchemicalinteractions[6–8],butalsoontheelectrostaticinteractionsbetweenthem[5,9–13].Whenthecarrierparticlesandproteinarepresentedinliquidphase,theydevelopanelectricchargeduetotheirioniccharacteristics,andeachparticleissur-roundedbya?xedlayerofoppositelychargedions(Sternlayer),whichisenvelopedbyadiffuselayerwithionsofdifferentpolar-

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