生物分離工程-雙語版-4知識講解

生物分離工程-雙語版-4IintroductionProteininterfacialadsorptionLowproteinconcentration(nobarrier)Highsurfaceproteinconcentration(barrier)AelectrostaticBstericCosmoticeffectProteinsadsorptiononGas-liquidinterface(1)enzymeadsorbedunfoldedpartiallyactiveorinactivestateasrigidfilm(2)proteinsunfoldingdependsonconformationalstability(3)interfacesareprimarilyresponsibleforproteininactivation(4)Shearassociateddamageisseverewhengas-liquidinterfacepresentApumpBcentrifugesCultrafiltration(5)proteinsonadsorptionatfluidinterfacesundergoachangefromtheirglobularconfigurationtoanextendedchainstructure(6)changeofproteinstructureleadtoactivitychangesProteinsadsorption1proteinmoleculesinaqueoussolutionhaveahydrophobicinteriorandahydrophilicexterior2structuralperturbation-rearrangementsinvolvingchangesininteratomicdistanceaffecttheintermolecularvanderwaalsinteraction3chargegroupsintheapolarinterioroftheproteinmoleculetendtoformionpairs.4intramolecularpeptidehydrogenbondsaremorefavorablethanpeptide-waterinteraction5thevariationsinthehydrogenbonding,ionpairing,hydrophobicinteractionaffecttherotationalmotionProteinsadsorption6Increaseofentropyoriginatingfromthedehydrationoftheinterface-surface,anddehydrationofstructuralchangesarethedrivingforce.7pHandshakingsignificantlyaffecttherateoftransportofproteintointerface.8Surfactantsoccupythesurfaceandpreventtheunfoldinganddenaturationofprotein.9ProteinsadsorbedexistmultiplebindingmodestatesAweaklyandtightlyboundproteinsBNotallofadsorbedproteinisremovedunderoneoraspecifiedsetofconditionsIIReactionandinactivationatliquid-liquidinterfaces1lipid-waterinterfacesAInhibitoryproteinpreventsthelipasefrombindingBSerumalbuminandβ-lactoglobulinresultinlipaseactivitylossof93and92%CDifferentconformationalstatesexhibitdifferentactivitiesandactivitiesofthisproteincouldberegulated.BAqueoustwo-phasesystems(1)Moderateselectivity(2)highyield(3)Biocompatibility(4)amenabilitytoscale-up(5)ElectrostaticinteractionplayasignificantpartinproteinadsorptionandinteractionattheinterfaceExample4.1PEG-FeSO4-water,PEG-Na2SO4-water(1)PEG-saltsystemareattractivecomparedwithPEG-dextransystem(2)PEGconcentrationlevelislowinsalt-richphaseArecoveryofbiomoleculesiseasierfromsalt-richphaseusingultrafiltrationBlimitedPEGlossReversedmicelles(1)Surfactantaggregateinorganicsolvent(2)Proteincanbeinducedtomovefrombulkaqueousphaseintomicelle-containingorganicsolventandviceversabymanipulationofpH,ionicstrength,andsurfactantconcentration.(3)Proteintransferisdominatedbyelectrostaticinteractions(4)SelectivelyseparatingproteinExample4.2interfacialtransportprocesses（1）Transferofα-chymotrypsinandcytochromeC（2）pHandsaltconcentrationinfluencebothforwardandbacktransferrateofprotein（3）Electrostaticsplayasignificantrole（4）Chargeinteractionwillproduceasignificantelectrostaticforceatinterface.（5）Proteinforwardtransferfasterthreeordersofmagnitudethandoesforwardtransferofsmallmolecules（6）ExtractionkineticsiscontrolledbyconvectivetransportExample4.2interfacialtransportprocessesMechanismsuggested:1Ruptureoforganicsolventfilmbetweenreversedmicelleandtheinterface2ReversedmicellemaydischargeitscontentstoaqueousphasebyAosmoticpressuredifference(forwater)Bcoalescence聚合IIIReactionandinactivationatgas-liquidinterfaces(1)Adsorptionofproteinsisimportantfortheoryandpractice(2)Proteinfilmsassistinstabilityoffoamsandemulsions(3)Transportofdissolvedgasesinfermentationbroth(4)FoamfractionationandfrothflotationAshearandagitation(1)stirringintroduceairintoaproteinsolution(2)enzymeswithsulfhydryl(SH)groupeasilyinactivatedbyoxidationofdisulfides(3)inactivationofhumanhemoglobinAandSatair-waterinterfaceAHemoglobinA:normalhumanBHemoglobinS:asickleredcelldiseasebprecipitatedwhenshakingcshakinginducesbubbleformation,agitationwithoutbubbleformationdgreaterareapermoleculeresultinagreaterunfolding(S8000A2,A5000A2Air-liquidsurfaceeffectplaymajorrole

inproteininactivation1Inactivationphenomena(1)unfolded(2)aggregate(3)precipitated2Shakingeffect(1)Filmformscreatesabarriertodiffusionwithoutshaking(2)Precipitatedfilmmaterialisremovedfromtheinterface,moreproteintobeadsorbedandprecipitatedwithshaking3Shearrateplaysanimportantroleininactivationofproteinattheair-waterinterface.4EnzymelostactivityduetodenaturationoftheircatalyticsitesbymechanicalshearExample4.3kineticsandmechanismofshearinactivationoflipaseShearingexperiment1Goodmixingisessential,butmixingproducesshear2Lipaseinactivationinvolvedinshear-inducedinterfaceeffect(1)shearincreaserateofadsorptionatair-waterinterface(2)splitsuporchangeitsconformation,unfold,orcoagulate.(3)shearassistininterfaceinactivationbyreplacingoldmoleculesatinterfaceandinturninactivatedbyinterfacetension3polypropyleneglycol(PPGantifoamagent)significantlydecreaselipasedenaturationAbindsdirectlytolipasetoprotectitsstructurefrominactivationBdecreasesthesurfacetensionandprotectslipasefrominactivationExample4.3kineticsandmechanismofshearinactivationoflipase4denaturationratecanbereducedtoverylowlevelbyadditionofsmallamountofsurfactantsorPEGormethylcellulose5denaturedβ-lactoglobulincanberenaturedbydissolvingitindiluteacid6surfactantpreferentiallyadsorbedoccupythesurfaceandpreventtheunfoldinganddenaturation.BtechniquesforproteinadsorptionTechniquestostudyadsorptionofproteinatinterfaces(1)filmbalance(2)pendantdropmethods(3)surfacetensionreduction(4)ellipsometry橢圓光度法(5)radiotracermethodAdirectlyquantifyproteinconcentrationatgas-liquidinterfaceBmoreradiationamountfrominterfacethanbulksolutionChighspecificandsensitiveDdoesnotdisturbadsorbingproteinsolutionEradiolabelingcouldalterconformationorhydrophobicityofmoleculesFcommonmethodofradiolabelingisaccetylationofterminalamineandlysineresiduesGremoveschargefromderivatizedresiduesandincreasehydrophobicityofproteinExample4.4radiotracertechniqueadsorptionofreductivelymethylatedlysozymetoair-waterinterface(1)proteinwasradiolableledbyreductivemethylationofterminalamineandlysineresidues(2)obtainanadsorptionisothermforlysozymemeasuredoverarangespanningsixordersofmagnitudeofbulkproteinconcentration(3)AdsorptionmechanismAbelowacriticalconcentration:orientation,untilsaturatedmonolayerBatthecriticalconcentration,end-onorientationCabovecriticalconcentration,saturatedmonolayerisattachedatinterface,secondlayerormultilayeroccurs(4)isothermexhibitsmonolayersaturationatlowbulkproteinconcentration(5)lysozymemoleculeinfirstlayerdonotexchangesignificantlywithlysozymeinbulksolutionKineticmodeldevelopedBelowcriticalconcentrationofproteininbulksolutionCmodelforproteinadsorptionModelexplanation1unsuccessfulforsomeproteinssuchasβ-lactoglobulin,α-lactalbumin,andBSA2somemodificationhavebeensuggested3modelissimplicity,butstillobtainpreciseandmoreextensivedata.4morecomplexmodelstwoadsorptionlayersAfirstlayeratinterfaceBsecondlayerfromadsorptionofproteinontothefirstlayerCgoodfitforadsorptionofβ-casein,BSA,andlysozymePeptidesadsorptionatinterfaces(1)peptideadsorptionisimportantAliquid-solidBair-water-liquid(2)peptide-hormonedrugs(lowdosesmakeinfluenceofadsorptionsignificant(3)usingpeptidesasmodelsubstanceforadsorptionstudiesisimportantAAVP(argininevasopressin加壓素）andd-AVP(desamino-8-argininevasopressin)(4)moresuchstudiesareurgentlyrequiredduetomedicalrelevanceNewwordsandvocabulariesImplants植入α-helixEnthalpy熱焓Entropy熵Patches片Perturbation動搖，混亂Ionpairs離子對Plethora過剩，過多Formidable艱難的，可怕的Formatedehydrogenase甲酸脫氫酶NewwordsandvocabulariesAcrylic丙烯酸的Falloff下降，跌落CytochromeC細胞色素CCoalescence合并，聯(lián)合Merge吞沒，融合Coagulate凝結(jié)PolypropyleneglycolPPGpendant懸掛，下垂Integer整數(shù)Temporal時間的DFoamfractionationExample4.5(1)Foam:madeupofgasbubblesdispersedinliquid(2)Foamstabilizedbysurface-activeagents(3)proteinaresurfactantandadsorbedoninterfaceofliquid-solid(4)parametersaffectingfoamingbehaviorApHBtemperatureCsaltsDsugarsElipidsFcolumnheightGcolumndiameter(5)conditionstoproducedorcollapsefoamaremildIVReactionandinactivationatliquid-solidinterfaceProteinadsorptionatsolid-liquidinterface(1)biofouling(2)thrombosis(3)immunologicreactiononsolidsupport(4)chromatographyseparation(5)cellculture(6)proteindrugdeliverycontactwithpolymermaterialsAQuantitativeaspectsandconformationalchange(1)quantitativeaspectsofproteinadsorption(2)proteinconformationandorientationinadsorbedlayer(3)mechanismsinvolvedatthesolid-liquidinterface(4)compositionandconformationalchangeatinterface(5)proteinscanbedisplacedbyotherproteinswithdifferentcharacteristicsAQuantitativeaspectsandconformationalchange(6)proteinadsorptiondependsonbothproteinandsurfaceAintrinsicproteinadsorptionkineticsBchemicalequilibriumCflowofsolutionpasttheadsorbingsurface(7)absenceoftechniquesfordeterminingthefinethree-dimensionalstructure(8)drivingforceforadsorptionmainlyatributedtoentropicgains(9)minimizeproteinadsorptionbymodifyingpolymerparticles(10)characterizetheconformationofproteinadsorbed-desorbedfrompolymerparticlesExample4.6conformationalchangeofproteinsadsorbedonsmallparticles(1)Proteinsadsorptionandtheirconformationchangedeterminingbiologicalprocess-interfacereactionwhenpolymericmaterialincontactwithbiologicalfluid(2)proteinadsorptiononpolystyreneparticlescanbemodulatedbycoatingparticles(3)proteinsunfoldedwhenadsorbedonuntreatedpolystyreneparticles(4)directevidenceforconformationalchange(5)resistantproteinadsorptionmaterialsmaybedevelopedExample4.7influenceofsurfacehydrophobicityontheconformationalchangesofadsorbedfibrinogenKineticandadsorptionamountoffibrinogen(1)StudymethodAfouriertransforminfraredspectroscopyandattenuatedtotalreflection(FTIR-ATR)Btotalinternalreflectionfluorescence(TIRF)(2)StudymaterialAgermaniumBpoly-hydroxylmethacrylateCpolystyreneDsilica(3)ResultsAextentofconformationalchangesisrelatedtosurfacehydrophobicityBadsorptioninducesconformationalchangesBadsorption-desorptionkineticstheory(1)kineticscontrolthedynamicbehaviorofmoleculesatinterface.(2)kineticstypesanalysisimportanceAproteinsadsorption-desorptionBadsorptionofsyntheticpolymeroncolloidalparticlesCmanufacturingselfassemblymonolayersormultilayersDrelaxationoffoams(composedofsurfactant-stabilizedbubbles)(3)attractiveness(howquicklymoleculesinsolutionareadsorbedonsurface(4)retentivenessdurationsurfaceholdsontotheadsorbedmoleculepriortodesorptionBadsorption-desorptionkineticstheory(5)optimizesurface-bulksystemintermsofweakorstrongsystemstohelpminimizedenarurationofprotein(6)differentproperitesofproteinsaffecttheiradsorptionatsolid-liquidinterfaceAmoleculesizeBchargeChydrophobicityofproteinDflexibilityofproteins(7)structuraladaptabilityofproteinsnaturallyaffecttheiradsorptionbehaviorExample4.8adsorptionbehaviorofdifferentproteins1ProteinsmaterialARibonucleaseABCytochromeCCLysozymeDα-lactalbuminEBSA2PolymermaterialsAPolystyreneBStyrene-2-hydroxyethylmethacrylateCSilicaDLateralinteractionExample4.8adsorptionbehaviorofdifferentproteins3ConditionsApHBIonicstrength4ProteinspropertiesAFlexibilityBHydrophobicitiesCIsoelectricpointsDLateralinteractionResults1hydrophobicinteractionforprotein-hydrophobicsurfaceinteractionarestrongerthanelectricstaticrepulsion2electricstaticinteractionbetweenprotein-hydrophilicsurfacedominate3largerproteinmoleculesshowmaxmiunadsorptionaroundisoelectricpoint4proteinsinteractionaffectedbyanumberoffactorsExample4.9Drivingforcesofsavinaseadsorbedonsolid-liquidinterfaceFoursetofinteractioninvolvedinproteinsadsorption1Protein-surfaceinteraction(netchargeofsurfaceandproteinismajorinteraction2Interfacedehydration(dehydrationofhydrophobicinterfacepromotesproteinadsorption,hydrophilicinterfaceopposesit)3ahydrophobicsurfaceandelectrostaticrepulsionpromotesunfoldingandleadstohighersurfaceareacontactofprotein4lateralinteractionisresistedbyelectrostaticrepulsionDrivingforcesofsavinaseadsorbedonpolystyreneandonglassAelectrostaticinteractionsarethemajordrivingforcessolid-waterinterfaceBdehydrationathydrophobicinterfaceandlateralinteractionaresmallcontributionCtheenzymeadsorbsinitsnativestate,withoutunfoldingAdsorptionoflysozymeandα-lactolbuminatinterface1PolymermaterialANegativelychargedpolystyrene(PS-)BVariablychargedhematite(α-Fe2O3)2ForceandsubprocessofinterfaceadsorptionASubprocessesleadingtoanincreaseinentropyprovideamajordrivingforceforproteinadsorptionBDehydrationofsorbentandproteinsurfaceismajorentropycontributionsCBothlysozymeandα-lactolbuminexhibitedasignificantamountofdenaturationonPS-

Dα-lactolbuminisalmostcompletelydenaturedonhydrophilicα-Fe2O3

ELysozymelosesonlyafractionofitsactivityduetohighstructurestabilityExample4.10Adsorptionoffungallipaselipolaseatsolid-liquidinterface1LipolaseextracellularlipasefromthermophilicfungusUsage:removefattysoilfromclothesindetergents2Lipase:catalyzethehydrolysisandformationofesterbonds3ActivitysignificantlyincreaseataninterfaceAhighconcentrationofsubstrateBabetterorientationandconformationalchangeCsurfaceactivereactionproductsformedatinterfacealsoinfluencesubsequentbindingDdelicatebalancebetweensurfacehydrationandelectricstaticinteractionsdeterminesadsorptionoflipaseonsolid-liquidinterfaceElateralenzyme-enzymerepulsionreactioninfluencestheplateauvalueLipolaseadsorbsasahardprotein,notunfoldingVConclusions(1)1Itisnecessarytounderstandproteinsadsorptionatdifferenttypesofinterface(G-L,L-L,L-S)2Proteinshaveathermodymamictendencytodiffuseandreactattheinterface.3Significantandsubtlerearrangementwillbehappenasproteinsadsorbandreactatinterface4Adsorbedmoleculemaycontinuouslychangeitsconfiguration-conformationatinterfaceVConcl