蛋白质工程-完整版

Membrane-basedtechniquesfortheseparationandpurificationofProteins,Membrane-basedtechniquesfortheseparationandpurificationofproteins,Introduction,Pressure-drivenmembranetechnologies,Membranechromatography,Electrophoreticmembranecontactor,Integratedmembranetechnologies,CONTENTS,Conclusions,1.Introduction,Definition,Advantage,Application,Amembranecanbedescribedasaninterphase,usuallyheterogeneous,actingasabarriertotheowofmolecularandionicspeciespresentintheliquidsand/orvaporscontactingthetwosurfaces.,Advantage:(1)selectivetransport,(2)efcientseparation,(3)notrequireadditives,(4)lessenergyconsumption.,Application:(1)demineralization,(2)desalination/puricationofwater,(3)bioseparationoffermentationproducts(4)milkfractionation,(5)decaidicationoffruitjuices.,Type:(1)microltration(MF),(2)clarication,sterileltrationandultraltration(UF),(3)Nanoltration(NF),(4)High-performancetangentialowltration(HPTFF).,Type,Definition,Disadvantage:(a)limitationoftheprocessstreamforrelativelylowconductivityoffeedstream,(b)ahigh-energyrequirement,(c)substantialheatproduction,and(d)changesintheprocessfeedduetoreactionattheelectrodePESiswidelyusedUFmembranematerial,becauseofitshighrigidity,creepresistance,goodthermalanddimensionalstabilities.OthertypesofpolymericUFmembranessuchaspolyacrylonitrilemembrane,regeneratedcellulosemembrane,celluloseacetatemembraneandceramicmembranes.,2.1.Proteinsseparation/puricationbyMF,2.2.roteinseparationbyUF,2.3.AdvancedUFtechniquesforproteinsseparations,2.4.Proteinseparation/puricationbyNF,2.5.MembranefoulingduringproteinseparationbyUFandMF,2.1.Proteinsseparation/puricationbyMF,ModulecongurationofMFincludehollow-ber,tubular,atplate,spiral-woundandrotatingdevices.Thetwostandardmodesofoperationaredead-endandcross-owcongurationsareshowninFig.2.Thesedevicesshowsignicantincreasesinproteintransmissionandcapacity.,MFiswidelyusedfortheseparation,puricationandclarifyingofprotein-containingsolutions,e.g.fortherecoveryofextracellularproteinsproducedviafermentationandfortheremovalofbacteriaandvirusesinthenalformulationoftherapeuticproteins.,2.1.1.AdvancedMFunderelectriceld,Mucheffortisstillbeingdevotedtodevelopingnewmembranemoduleswithimprovedmass-transfercharacteristicsforUFandMFprocesses.Electricallyenhancedmembraneltration(EMF)isanadvancedtechnique,whichconsistsinsuperimposinganelectricaleldtoaconventionalembraneltrationunit.InEMF,theelectricaleldactsasanadditionaldrivingforcetothetransmembranepressure.,Advangtage:(1)selectivityenhancement,(2)improveproteinsolutionspermeationux,(3)reducethesurfacelayerofamembrane,2.2.ProteinseparationbyUF,UFmembranes,basedonvarietyofsyntheticpolymers,havehighthermalstability,chemicalresistivity,andrestrictedtheuseoffairlyharshcleaningchemicals.,Modules:(1)Hollowber,(2)at-sheetcassettes,(3)spiral-woundcartridges,(4)tubularmodules,(5)enhancedmasstransferdevices,Proteinfractionationisrapidlybecomingmoreselectivethroughimprovementsinmembraneandmoduledesign.,2.3.1.ProteinseparationusingchargedUFmembranes,ChargedUFmembraneseparationprocessinvolvedbothsizeandchargebasedexclusionratherthansimplysize-basedseparationofproteinmolecules,asinthecaseofUF.,Factor:(1)pHvalues,(2)ionicstrengths,(3)permeateux,(4)andsystemhydrodynamics.Advantange:extremelylowfoulingduetoelectrostaticrepulsion.,Material:(1)polyethersulfone,(2)polysulfone,(2)celluloseacetate,(3)regeneratedcellulose,(4)poly(ethyleneglycol)(PEG),(5)poly(furfuralalcohol)(PFA).,2.3.2.UFinthepresenceofelectriceld(electro-ultraltration),TheuseofelectriceldinUFgoesbacktotherststudycarriedoutbyBechholdbyimposingelectriceldinUFandutilizedacombinationofelectroosmosisandelectrophoresistopurifycolloidsinanapparatushecalledanelectro-ultraltration(EUF).,EUFisaneffectivemethodtodecreasegellayerformationonthemembranesurfaceandtoincreasetheltrationux,owingtoelectrokineticphenomenasuchaselectrophoresisandelectroosmosis,Factor:(1)electriceld,(2)solventow,Advantage:(1)enhancetheltrationrate,(2)increasestheseparationefciencyoftheUFofproteins.,Forthisreason,attentionhasbeendirectedtotheuseofpulsedelectricelds.Advantage:(1)lessenergy,(2)higherux,(3)reducingfouling,(4)restoringhighpermeationrate.,Disadvantage:(1)limitationoftheprocessstreamforrelativelylowconductivityoffeedstream,(2)ahigh-energyrequirement,(3)substantialheatproduction,and(4)changesintheprocessfeedduetoreactionattheelectrode.,Theapplicationoftheelectricpulseinthecleaningmembranesurfacewasaneffectivemeansinreducingfoulingandrestoringhighpermeationrate.,2.3.3UFinthepresenceofultrasonicfield,concentrationpolarizationandmembranefoulingleadtothedeclinationofpermeatesflux.,concentrationpolarizationreason:aconcentrationgradientoftheretainedcomponentsisformedonornearthemembranesurface.,Ultrasoundgeneratesacousticstreamingandcavitationbubblesinaliquidmedium,Cavitationbubblecausesmicrostreaming,microstreamers,microjets,andshockwaves.,membranefoulingreason:accumulationofproteinsdrawntowardfilteringsurfacebyconvectiveflowoffiltratethroughthemembrane.,VariousmethodshavebeenusedtoreducethenegativeeffectsofconcentrationpolarizationandfoulingforenhancethepermeatefluxandmembraneseparationefficiencysuchasUltrasonicphysicaleffectsandsonochemicaleffects.,Releasefromaparticle-fouledsurfacemechanism,Higherfrequencyultrasoundtendstohavehigherenergyabsorptionandthusgreateracousticstreamingflowratesthanlowerfrequenciesforthesamepowerintensity,higherpowerintensitiesleadtogreateracousticstreamingflowrates.,Thismechanismcausesbulkwatermovementtowardandawayfromthemembranecakelayer,withvelocitygradientsnearthecakelayerthatmayscourparticlesfromthesurface.,TheeffectofultrasonicwaveonseparationperformanceofproteinmixturebyUF,Theeffectofultrasoundonthefluxandsoluterejectionincross-flowUFofBSA-lysozymebinaryproteinmixtureusingPESmembraneultrasonicwavenotonlyenhancedtheUFfluxbutalsoincreasedthelysozymerejection.,ultrasoundwave(25kHzand240W)resultedinanincreaseofUFfluxby135%and120%withPESmembraneatpH:11intheupwardanddownwardmodes,respectively,incontrasttothecaseofwithoutanyultrasound.,MuralidharaandTarleton:electricandultrasonicfieldscanreducemembranefoulingandinturnofenhancedflux.additionWakemanandWilliams:Bothelectricalandultrasonicfieldsreducedthefoulingwhenappliedindividually,buttheextentofimprovementbytheultrasonicfieldcouldbeminimal.,Notice:theeffectivenessdependsonmanyfactors,suchasorientationandpositionofultrasonicfield,ultrasonicfrequencyandpower,ultrasonicradiationangle,positionofultrasonicvibrationplateinthemembranemodule,membranematerial,membranhousing,operatingpressureandthefoulingmaterial.Reusit:ultrasoniccavitation,acousticstreaming,ultrasonicinducedvibrationofmembraneandultrasonicheatingwasthemaincauses.,2.3.4.High-performancetangentialflowfiltration,HPTFF:anemergingtechnology、similarsize（Conventionalten-foldinsize，nowlessthanthreefoldinsize）.,Optimumselectivityandthroughput，enhancedthroughmoduledesignandprocessconfigurations.,Sievingbehaviorimpact:OptimizationsofbufferpHandionicstrength.,Atwodimensionalunitoperation,proteinconcentration,purificationandbufferexchangecanbeaccomplishedinasingleunitoperation.,Somestrategiestoachievehighresolutionseparations,(1)properchoiceofpHandionicstrengthtomaximizedifferencesinthehydrodynamicvolumeoftheproductandimpurity.,(2)useofelectricallychargedmembranestoenhancetheretentionoflikechargedproteins.,(3)operationinthepressure-dependentregimetomaximizetheselectivity.,(4)useofadia-filtrationmodetowashimpuritiesthroughthemembrane.,ComparisonofflowandpressureprofilesforconventionalTFFmoduleandco-flowarrangement,2.4.Proteinseparation/purificationbyNF,NF：suitablecut-offoftheNFmembranesandtheelectrochemicaleffects.,1、Negativelychargedmembraneshavebeenappliedtoenrichcationicpeptideswithantibacterialpropertiesfromcheesewhey.,2、Pouliot：theseparationofpeptidesfromtryptichydrolysatesofwheyproteinswithchargedUF/NFmembranes.,3、VariationinpHandtheionicstrength.,2.5.MembranefoulingduringproteinseparationbyUFandMF,Membranefouling：adsorptiononmembranesurfacesignificantlyincreaseshydraulicresistancetoflow,reducedfiltrationfluxrateandinducedunfavorableeffectonefficiencyandeconomicsofproteinrecoveryprocesses.,Foulingforms：（i）Theformationofagellayerduetoconcentrationpolarization.,(ii)Adsorptionofspeciesonthemembranesurfaceandinsidetheporestructure.,(iii)Depositionandporeblockingaftertheformationofproteinaggregatesduetodenaturation.,HoandZydney：developedacombinedmembranefoulingmodelporeblockageandcakefiltrationtodescribefluxdecline.,Themodelshowsasmoothtransitionfromporeblockagetocakefiltration,andisingoodagreementwithfluxdeclinedataobtainedduringbovineserumalbuminfiltrationusingpolycarbonatetracketchedmembranes.,Butinternalfoulingwascompletelyneglected.,welcometousethesepowerpointtemplates,NewContentdesign,10yearsexperience,A,B,Microsieves,theadvantageisthelargerpermeateflux,whichallowslow-pressureoperationandsavingsintheoperationalcostsandanotheradvantageofmicrosievesoverMFmembranesistheirstructuraldesign,averythinselectivelayerandperfectlyshapedstraightpores.,Surfacemodification,suchascoating,surfacegraftpolymerization,andchemicalmodification.,Nakao:studiedthatproteinfoulingduringUFwasentirelyduetotheformationofasecondary(gel)layerontheuppersurfaceofthemembrane.,Jiang.synthesizedpegylatedPESviaareactionofsulfonatedPESwitholigomericpoly(ethyleneglycol)(PEG).ThemodifiedmembranesshowedsuperiorresistancetoBSAadsorptionincomparewithunmodifiedcounterparts.,3.Membranechromatography,TITLE,ligandmoleculesareimmobilizedontheporoussurfaceoftheembeddedparticlesandthemixturecontainingtheproteinofinterestispassedthroughtheaffinitymembrane.,welcometousethesepowerpointtemplates,NewContentdesign,10yearsexperience,A,B,Threetypesofmembraneadsorbers:flatsheet,hollowfiberandradialflow,Flat-sheetmembraneadsorbers,theliquidwasusuallyintroducedtothemembranesurface.Stacksofseveralflatsheetswerehousedwithinmembranemodules.,Ahollow-fibermembraneadsorberusuallyconsistsofabundleofseveralhundredfiberspottedtogetherwithinamoduleinashellandtubeheat-exchanger-typeconfiguration.,Radialflowadsorberswereclaimedtobesuitableforlarge-scaleapplications.,welcometousethesepowerpointtemplates,NewContentdesign,10yearsexperience,A,B,3.1.Microporousmaterialsformembranechromatography,3.2.Affinityion-exchangematerialsformembranechromatography,3.3.Applicationofmembranechromatographyforproteinseparation,Theusesofion-exchangeandaffinityinteractionsaremorewidelyreported,butonlysmallworkhasbeendoneonhydrophobicinteractionandreversed-phasebasedmembranechromatographyofproteins.,Theligands（配体）usedforaffinity-membranechromatographycanbebroadlyclassifiedintofourtypes:iimmunoaffinityligands,iiprotein,iiilow-molecular-massligands,ivotherligands.,Lysozyme（溶菌酶）separationfromeggwhitewasachievedefficientlyusingmacroporouschitinmembrane,with98%purity.,Thiophilicmembranes（嗜硫膜）forthepurificationofmonoclonalantibodies,Hollow-fibermembranes（中空纤维膜）fortheseparationofIG,Stronganion-exchangemembranesforreductionofendotoxin,Ion-exchangemembranesfortheisolationofantibacterialpeptidesfromlactoferrin,Cation-exchangemembranesforthepurificationofalphaviruses,AffinitymembranesfortheseparationofMBPfusionproteins,4.Electrophoreticmembranecontactorfortheseparationofproteins,Differentstudiesweredevotedtofindouttheoperatingmodesforscale-upofelectrophoreticseparations.Oneofthemiscontinuousflowelectrophoresis(CFE)（连续流动电泳）.,Thelimitationswereintermsofproductioncapacityorproductivity,demonstratedandithadastrongrelationshipbetweenresolutionandproductivity.,Itwasfoundthattheproductivitycouldnotbeincreasedoveracertainlimit,typicallyaboutfewmilligramsperhour.,Toovercometheselimitations,electro-membraneoperationsofferedthepossibilitytoincreasetheproductivitywithoutcompromisingfromseparationefficiency.Themostcommonelectromembraneoperationiselectrodialysis(ED)（电渗析）inwhichion-exchangemembranesareused.,Inthatcase,theporousmembraneactsasacontactorandtheseparationisachievedwithrespecttothedifferencebetweenthemassflowratesofthespecies.,Electrophoreticmembranecontactorwasdevelopedfortheseparationofbinarymixtureofproteinsbyusingion-exchangeandultrafiltermembranes.,5.Integratedmembranetechnologiesforproteinseparation,Membraneprocessforfoodanddairyindustry,Milkanddairyindustry,Theintegrationofmembraneshasbeenimplementedthroughoutthemilkanddairyprocessingchains-milkreception,cheesemaking,wheyproteinconcentration,fractionationofproteinhydrolysates,wastestreampurificationandeffluentsrecyclingandTreatment.,Foodandbeverageindustries,Recentindustrialapplicationshavebeendevelopedforfruit,vegetableandsugarjuicesandbeverages(vegetableproteins,beer,andwine).Furtherintegrationofmembraneoperationsweredesignedinsuchawaythatateachprocessingstep,endproducts,co-productsandwastesaregivenevenattention.,A,B,Membraneprocessforfoodanddairyindustry,Membraneprocess,Removalofbacteriaandsporesfromskimmilk(coldpasteurization),MFreducestheamountofbacteria,sporeswithoutaffectingthetasteofmilk,andprovideslongershelflifethanpasteurization.Besidestheproductionofconsumptionmilkwithextendedshelflife（保质期）,thismethodcanbeusedaspretreatmentofskimmilk（脱脂牛奶）fortheproductionofrawmilkcheesesandthereductionofsporesinacidcheesemilk.,Arecentdevelopmentisthemicrosieves,whichwasmadewithmicro-machiningtechnology.Withmicrosieves,ahighreductionofbacteriacanbeachievedatlowtransmembranepressure,sincethesemembraneshaveveryhighpermeabilities.Hence,theuseofmicrosievesseemsverypromising.,Recoveryofserumproteinsan