酶的分离工程酶的分离工程PPTPPT课课件件ObjectivesPurityStableCostTimePurificationtypicallyinvolvesthreesteps1)Preparationofacrudeextractfromharvestedcells2)Fractionation:Separationofamixtureofproteinsintovariousfractionsaccordingtosomeproperty(e.g.size,charge,solubility)3)SeparationofproteinfromsolventsandconcentrationUnit1PreparationofCrudeEnzymesEndoenzyme:intracellularMostenzymesofthemetabolicpathways.Exoenzyme:extracellularBreakdown(hydrolyze)largefoodmoleculesorharmfulchemicals.Example:cellulase,amylase,penicillinase.Solid/LiquidSeparation-centrifugation and filtrationWhenharvestingbrothcultures,howarecellsseparatedfromthebroth?Decanter CentrifugeClarifiedliquidRotatingBowlRotatingscrollFrameFilterHowtoimprovefiltervelocity?1）FlocculationandAgglomeration2） Decreaseviscosity3）FilteraidCellDisruptionThemaincomponentofcellwalllBacteria:PeptidoglycanlYeast：Dextran，Mannose，ProteinlMycelialfungus:Chitin,Dextranl Gram Positive Bacterial Cell WallGram Negative Cell WallFungusCellWallMechanicalmethodsGrinding(inliquidnitrogen,ballmill）DrywayHomogenization（mortar,homogenizer)-WetwayPhysicalmethodstemperaturedifference（ freezingandthawing）pressuredifference( osmoticshock)ultrasonicationChemicaltreatmentorganicsolventsdetergents：TritonX-100，Tween（usedifenzymeisinlipidmembrane）EnzymelysisautolysisextraenzymeWaystobreakcellBeadMillCascadingbeadsCellsbeingdisruptedRollingbeadsSonicatorSonicatorSonicatorSonicatorDisruptstissuebycreatingvibrationswhichcausemechanicalshearingofthecellwall.Afterbreakingthecell1)Keeptemperaturelow2)Purifyassoonaspossible3)Avoidoxidation4)AvoidcontaminationCoolingandproteaseinhibitionareimportanttorecovertheenzyme! EnzymeExtractionFromplantandanimaltissue.Toachievemaximumsolubilityandactivityoftheenzyme. ExtractmethodsSolventorSolutionextracttargetsaltingliquid0.020.5mol/LNaClsolutionacidsolutionpH26aqueoussolutionalkalisolutionpH812aqueoussolutionorganicsolventwater-miscibleorganicsolventMethodsforExtractionofEnzymesUnit2MethodsofPurificationCentrifugationPreparativecentrifugationAnalyticalcentrifugationPreparativecentrifugationCollectmaterialcellsprecipitatedmacromoleculesSubcellularfractionationAnalyticalCentrifugationSedimentationCoefficient(s)isthevelocityperFc,ors=v/2runitisSvedberg,where1S=10-13secRelativeCentrifugalForceand and RotationPerMinuteexpressedasxgravityRCF=Fc/Fg=2r/980=(rpm)/30RCF=1.11910-5(rpm)2rTheunitis“g”Speed(rpm)Importantnoticelowspeedcentrifuge 6000R.T.equilibriumhighspeedcentrifuge600025000 freezingequilibriumexactlyultraspeedcentrifuge25000freezingvacuumsystemequilibriumexactlyTypesofCentrifugesHow to use acentrifuge? EquilibriumSettempreatureSetrpmTiming CentrifugationMethodsDifferentialcentrifugationHighspeedSedimentationvelocitySedimentationequilibriumSuperhighspeed1 1）Differential Centrifugation Differential Centrifugation (Gravity Centrifugation)(Gravity Centrifugation)Separatesupernatantandpelletbymassanddensitypreparecelllysatesubjecttocentrifugationcentrifugalforcetime(gmin)tubesizeandshaperotoranglere-centrifugesupernatantProblemscontaminationlargeparticlescontaminatedwithsmallerparticlesresolutionparticlesofsimilarsizesnotseparatedvibrationsandconvectioncurrents2)SedimentationVelocityRateZonalps(大大)separatesprimarilybymasscommonmedia:sucrose3)SedimentationequilibriumIsodensitys（小）（小）ps时时, , V V0,0,样品顺离心力方向沉降样品顺离心力方向沉降 ps时时, , V V0.1M）,thechargedmoleculesarequicklyprecipitatedbecausetheexcessions(notboundtotheprotein)competewithproteinsforthesolvent.Salting-outeffect:ionstakeallwater,exposethenonpolarsurface;solubilitydecrease!PrecipitationProteinmoleculesaredehydratedbystrongsaltsolution.Thechargesofproteinmoleculesareneutralized.Atlowconcentrations,thepresenceofsaltstabilizesthevariouschargedgroupsonaproteinmolecule,thusattractingproteinintothesolutionandenhancingthesolubilityofprotein.Thisiscommonlyknownassalting-in.However,asthesaltconcentrationisincreased,apointofmaximumproteinsolubilityisusuallyreached.Furtherincreaseinthesaltconcentrationimpliesthatthereislessandlesswateravailabletosolubilizeprotein.Finally,proteinstartstoprecipitatewhentherearenotsufficientwatermoleculestointeractwithproteinmolecules.Thisphenomenonofproteinprecipitationinthepresenceofexcesssaltisknownassalting-out.Usedtoselectivelyprecipitateproteins,oftenwith(NH4)2SO4whichischeap,effective,doesnotdisturbstructureandisverysoluble.Saltingout(Ammoniumsulfateprecipitation)SaltconcentrationisindictedinPercentagesaturation（饱和度）（饱和度）Volumeofsaturated(NH4)2SO4SaturationratioTotalsolutionvolumeHowtoachievedesiredpercentageofammoniumsulfate？Add-saturatedsolution-drypowderHowtomakingX%solutionfromXo%solution?The effect of salt on different proteins may differ:Certain proteins precipitate from solution under conditions in which others remain quite soluble.Once the protein is precipitated (not denatured) can separate by centrifugation pellet can be redissolved in buffer for further purification Which protein will ppt first? (hydrophobic or hydrophilic?) FractionalsaltingoutDifferentproteinsprecipitateatdifferentsaltconcentration.serumglobulinalbumin(NH(NH4 4) )2 2SOSO4 450%saturationsaturatedprecipitateprecipitateSaltingoutcurvelprotein(mg)orenzymeactivity 10 20 30 40 50 60 70 80 90 100(NH(NH4 4) )2 2SOSO4 4 percentageofsaturated Inbrief,theproceduregoesasfollows:1)obtainproteinsolutionofinterest2)add(NH4)2SO4toachilled,stirringsolution3)allowtostirfor15-30minutes4)collectprecipitatedproteinbycentrifugation5) re-dissolvedinbufferforfurtherpurificationlImportantfactors:l1)ionicstrengthlS=solubilityoftheproteinKsKs：salt-specificconstant: : idealizedsolubility I:theionicstrengthofthesolutionlog S = - Klog S = - Ks s I I l2)pH: pIpIl3)temperaturellowionicstrength, ,T.proteinsolubilitylhighionicstrength, ,T.proteinsolubilityl4)proteinconcentration:moderate2.PrecipitationwithorganicsolventsDecreaseindielectricconstantlOrganicsolventdecreasesthewateractivityandthedielectricconstantofthesolution,whichthendecreasesthesolubilityoftheproteinandprecipitatesit.Commonorganicsolvent: acetone ethanol methanol，2proteinvolumelImportantfactors1)1)Temperature：low（0 0） Because.a.a.Someproteinsmightbedenaturedbyheatproduced.b.Increaseenzymeyield（T.,solubility）2) pH2) pH：pIpI3)3) Proteinconcentration:moderateSalting-inSalting-outOrganicsolventReagentsNaCl(monovalent)(NH4)2SO4(divalent)Acetone,ethanol,methanolRemarksThereverseprocessofsalting-inisnotsalting-out,itisthedialysisprocess.1)Non-polarproteinswillbeprecipitatedearlier.2)Proteinisverystablein(NH4)2SO4.1)Someproteinsmightbedenaturedbyheatproduced.2)Factorsfacilitateprecipitation:largerprotein,pHclosetoproteinpI.3)Lipophilicproteinmightbedissolvedmorereadily.Comparisonoftwomethods3.IsoelectricpointprecipitationlChangeinpH -EnzymesareleastsolubleatpI-DifferentenzymeshavedifferentpIlUsingmethodIt seldom be used alone（ often used toremoveundesiredprotein）.Because：ManyproteinshavesimilarpI.ProteinshavesomesolubilityatpI.4.Non-ionichydrophilicpolymers（Polyethyleneglycol（PEG）precipitation）Molecularweight:600020000RemarksPEGwillprecipitateswithoutdenaturing.Itsprecipitationeffectsisveryhigh.5.SelectivedenaturationAnegativemethodleavesthedesiredproteinactiveinsolution;heat;extremepH;organicsolvents;Relationshipbetweendenaturationandprecipitation?Casein(酪蛋白酪蛋白),denaturedinboilingmilk,willnotbeprecipitated.Proteinisnotdenaturedbysaltingout.Extraction Amethodtoseparatecompoundsbasedontheirrelativesolubilitiesintwodifferentimmiscibleliquids. Organicsolventwater1.Aqueoustwo-phaseextraction（ATPE） Specialcasedofliquid-liquidextractionTwotypesofaqueoustwo-phasesystems:Polymer-polymertwo-phasesystemle.g.:dextranandPEGPolymer-salttwo-phasesystemle.g.PEGandKClPEGdextransystemTheupperphaseisformedbythemorehydrophobicpolyethyleneglycol(PEG),whichisoflowerdensitythanthelowerphase,consistingofthemorehydrophilicanddenserdextransolution. Aqueous two-phase extractionBiphasicsystemMonophasicsystemDextran%w/wTielinesPEG%w/w Foreverysubstance,thereisacriticaltemperature(Tc)andpressure(Pc)abovewhichnoappliedpressurecanforcethesubstanceintoitsliquidphase.IfthetemperatureandpressureofasubstancearebothhigherthantheTcandPcforthatsubstance,thesubstanceisdefinedasasupercriticalfluid.WhatisaSupercriticalFluid?2.SupercriticalFluidExtraction（SFE）Supercritical Fluid ExtractionlSFcombinesdesirablepropertiesofgasesandliquidsSolubilityofliquidsPenetrationpowerofgasesSolvent(SF)SolutePhaseDensity(g/ml)Diffusioncoefficient(cm2/s)Viscosity(g/cm/s)Gas10-310-110-4SCF0.30.910-310-410-410-3Liquid110-510-2The Choice of the SFE SolventCarbondioxideisthemostcommonlyusedSCF,dueprimarilytoitslowcriticalparameters(31.1C,73.8bar),lowcostandnon-toxicity. lDensityofSFandsolubilityofasoluteinitcanbechangedinacontinuousmannerbychangeofpressureSupercriticalFluidExtractionProcess-flexibilitySFE: TypeslLiquid-SFextractionSimilartoliquid-liquidextractionExamples:lRemovalofalcoholfrombeerlSolid-SFextractionSimilartosolid-liquidextraction(leaching)Examples:lRemovalofcaffeinefromcoffeebeans3.ReversedmicelleextractionlReversedMicelles:surfaceactiveagentorganicsolvent Q: Whats the different between reversed micelle extraction and solvent extraction ? Filtrationa.Proteinsolutionthroughamembranewhichretainstheproteinofinterest.b.Thismethodislesslikelytocausedenaturation.membraneseparationDiffusionmembraneMicrofiltrationUltrafiltrationReverseosmosispressuremembraneElectrodialysis dialysisElectromembrane1.Diffusionmembrane（concentrationdifferencedrivenprocess）dialysis Dialysis tubing has pores with a specific molecular weight cut-off that allows smaller molecules (salt) to pass.Purposes:Reduce ionic strength of the solution.Concentrate protein sample.DialysisDialysis Typically,processinvolvesseveralchangesofbuffersothatthesaltconcentrationinthesampleisreducedtoacceptablelevel.Whathappensduringdialysis?Whyisdialysisanimportanttechniqueinproteinpurification?Q:Whyisbloodred?Howtotestify?2.Pressuremembrane（Pressuredifferencedrivenprocess）a.Microfiltration（MF）Microfiltrationisafiltrationprocesswhichremovescontaminantsfromafluid(liquid&gas)bypassagethroughamicroporousmembrane(0.1to10m).Someexamplesformicrofiltrationb. Ultrafiltration（UF）Usuallyusedtofurtherseparateanycontaminantsabletopassthroughthemicrofiltrationmembraneusingapressuregradient.SmallmoleculesarefilteredoutbypressureUsedforconcentratingproteinsAlternatively,centrifugationwithdialysismembrane超滤浓缩装置超滤浓缩装置 c. c. Reverseosmosis（RO） Osmosisisthe“movementofasolventthroughasemi-permeablemembraneintoasolutionofhighersoluteconcentration”.Theory of OsmosisFreshWaterSeaWaterH2OInitialConditionFreshWaterSeaWater(diluted)H2OEquilibriumH2OSemi-permeableMembraneFreshWaterSeaWaterH2OPressureReverseOsmosisRO Membrane Filter DetailIndustrial Applications of RO SystemslPurification of potable or “fresh” water sourceslDesalination of sea water diameterParticlesRecoveryPressureApplicationRFRF 2m 2mYeast , Yeast , mould、plant or animal cell, plant or animal cell, solidsnormal normal pressure pressure solid-liquid separationMFMF 0.20.22m2m Bacteria, Bacteria, dust et al.0.1MPa0.1MPadegermationUFUF20200.2m0.2mvirus, virus, biological macromolecules et al.0.1 0.1 0.7MPa0.7MPapurification of virus and biological macromoleculesRORO 20 20biological small molecules, salt et , salt et al.al.0.7 0.7 13MPa13MPaPreparation of high purity waterReverse Osmosis (RO)Nanofiltration (NF)Ultrafiltration (UF)Microfiltration (MF)Membrane Filtration3.Electromembrane（electricalpotentialdifferencedrivenprocess）Electro dialysis（ED）ElectroDialysis(ED)isamembraneprocess,duringwhichionsaretransportedthroughcation-oranion-selectivemembranes,undertheinfluenceofanelectricpotential.SomeExamplesRecoveryofmicroorganismfromfermentationbroth.Removingpyrogens(热原热原)from injection.ConcentrationofmilkExtractingeffectiveingredientsfromdomesticChineseherbs.结束结束