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1、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)Separationofproteinfromsolventsandconcentrat
2、ionUnit1PreparationofCrudeEnzymesEndoenzyme:intracellularMostenzymesofthemetabolicpathways.Exoenzyme:extracellularBreakdown(hydrolyze)largefoodmoleculesorharmfulchemicals.Example:cellulase,amylase,penicillinase.Solid/LiquidSeparation-centrifugation and filtrationWhenharvestingbrothcultures,howarecel
3、lsseparatedfromthebroth?Decanter CentrifugeClarifiedliquidRotatingBowlRotatingscrollFrameFilterHowtoimprovefiltervelocity?1）FlocculationandAgglomeration2） Decreaseviscosity3）FilteraidCellDisruptionThemaincomponentofcellwalllBacteria:PeptidoglycanlYeast：Dextran，Mannose，ProteinlMycelialfungus:Chitin,D
4、extranl Gram Positive Bacterial Cell WallGram Negative Cell WallFungusCellWallMechanicalmethodsGrinding(inliquidnitrogen,ballmill）DrywayHomogenization（mortar,homogenizer)-WetwayPhysicalmethodstemperaturedifference（ freezingandthawing）pressuredifference( osmoticshock)ultrasonicationChemicaltreatmento
5、rganicsolventsdetergents：TritonX-100，Tween（usedifenzymeisinlipidmembrane）EnzymelysisautolysisextraenzymeWaystobreakcellBeadMillCascadingbeadsCellsbeingdisruptedRollingbeadsSonicatorSonicatorSonicatorSonicatorDisruptstissuebycreatingvibrationswhichcausemechanicalshearingofthecellwall.Afterbreakingthe
6、cell1)Keeptemperaturelow2)Purifyassoonaspossible3)Avoidoxidation4)AvoidcontaminationCoolingandproteaseinhibitionareimportanttorecovertheenzyme! EnzymeExtractionFromplantandanimaltissue.Toachievemaximumsolubilityandactivityoftheenzyme. ExtractmethodsSolventorSolutionextracttargetsaltingliquid0.020.5m
7、ol/LNaClsolutionacidsolutionpH26aqueoussolutionalkalisolutionpH812aqueoussolutionorganicsolventwater-miscibleorganicsolventMethodsforExtractionofEnzymesUnit2MethodsofPurificationCentrifugationPreparativecentrifugationAnalyticalcentrifugationPreparativecentrifugationCollectmaterialcellsprecipitatedma
8、cromoleculesSubcellularfractionationAnalyticalCentrifugationSedimentationCoefficient(s)isthevelocityperFc,ors=v/2runitisSvedberg,where1S=10-13secRelativeCentrifugalForceand and RotationPerMinuteexpressedasxgravityRCF=Fc/Fg=2r/980=(rpm)/30RCF=1.11910-5(rpm)2rTheunitis“g”Speed(rpm)Importantnoticelowsp
9、eedcentrifuge 6000R.T.equilibriumhighspeedcentrifuge600025000 freezingequilibriumexactlyultraspeedcentrifuge25000freezingvacuumsystemequilibriumexactlyTypesofCentrifugesHow to use acentrifuge? EquilibriumSettempreatureSetrpmTiming CentrifugationMethodsDifferentialcentrifugationHighspeedSedimentation
10、velocitySedimentationequilibriumSuperhighspeed1 1）Differential Centrifugation Differential Centrifugation (Gravity Centrifugation)(Gravity Centrifugation)Separatesupernatantandpelletbymassanddensitypreparecelllysatesubjecttocentrifugationcentrifugalforcetime(gmin)tubesizeandshaperotoranglere-centrif
11、ugesupernatantProblemscontaminationlargeparticlescontaminatedwithsmallerparticlesresolutionparticlesofsimilarsizesnotseparatedvibrationsandconvectioncurrents2)SedimentationVelocityRateZonalps(大大)separatesprimarilybymasscommonmedia:sucrose3)SedimentationequilibriumIsodensitys（小）（小）ps时时, , V V0,0,样品顺离
12、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.Thecharg
13、esofproteinmoleculesareneutralized.Atlowconcentrations,thepresenceofsaltstabilizesthevariouschargedgroupsonaproteinmolecule,thusattractingproteinintothesolutionandenhancingthesolubilityofprotein.Thisiscommonlyknownassalting-in.However,asthesaltconcentrationisincreased,apointofmaximumproteinsolubilit
14、yisusuallyreached.Furtherincreaseinthesaltconcentrationimpliesthatthereislessandlesswateravailabletosolubilizeprotein.Finally,proteinstartstoprecipitatewhentherearenotsufficientwatermoleculestointeractwithproteinmolecules.Thisphenomenonofproteinprecipitationinthepresenceofexcesssaltisknownassalting-
15、out.Usedtoselectivelyprecipitateproteins,oftenwith(NH4)2SO4whichischeap,effective,doesnotdisturbstructureandisverysoluble.Saltingout(Ammoniumsulfateprecipitation)SaltconcentrationisindictedinPercentagesaturation（饱和度）（饱和度）Volumeofsaturated(NH4)2SO4SaturationratioTotalsolutionvolumeHowtoachievedesired
16、percentageofammoniumsulfate？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 se
17、parate 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%saturationsaturatedprecipitatepr
18、ecipitateSaltingoutcurvelprotein(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)collectprecipitatedproteinbycentri
19、fugation5) re-dissolvedinbufferforfurtherpurificationlImportantfactors:l1)ionicstrengthlS=solubilityoftheproteinKsKs：salt-specificconstant: : idealizedsolubility I:theionicstrengthofthesolutionlog S = - Klog S = - Ks s I I l2)pH: pIpIl3)temperaturellowionicstrength, ,T.proteinsolubilitylhighionicstr
20、ength, ,T.proteinsolubilityl4)proteinconcentration:moderate2.PrecipitationwithorganicsolventsDecreaseindielectricconstantlOrganicsolventdecreasesthewateractivityandthedielectricconstantofthesolution,whichthendecreasesthesolubilityoftheproteinandprecipitatesit.Commonorganicsolvent: acetone ethanol me
21、thanol，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,met
22、hanolRemarksThereverseprocessofsalting-inisnotsalting-out,itisthedialysisprocess.1)Non-polarproteinswillbeprecipitatedearlier.2)Proteinisverystablein(NH4)2SO4.1)Someproteinsmightbedenaturedbyheatproduced.2)Factorsfacilitateprecipitation:largerprotein,pHclosetoproteinpI.3)Lipophilicproteinmightbediss
23、olvedmorereadily.Comparisonoftwomethods3.IsoelectricpointprecipitationlChangeinpH -EnzymesareleastsolubleatpI-DifferentenzymeshavedifferentpIlUsingmethodIt seldom be used alone（ often used toremoveundesiredprotein）.Because：ManyproteinshavesimilarpI.ProteinshavesomesolubilityatpI.4.Non-ionichydrophil
24、icpolymers（Polyethyleneglycol（PEG）precipitation）Molecularweight:600020000RemarksPEGwillprecipitateswithoutdenaturing.Itsprecipitationeffectsisveryhigh.5.SelectivedenaturationAnegativemethodleavesthedesiredproteinactiveinsolution;heat;extremepH;organicsolvents;Relationshipbetweendenaturationandprecip
25、itation?Casein(酪蛋白酪蛋白),denaturedinboilingmilk,willnotbeprecipitated.Proteinisnotdenaturedbysaltingout.Extraction Amethodtoseparatecompoundsbasedontheirrelativesolubilitiesintwodifferentimmiscibleliquids. Organicsolventwater1.Aqueoustwo-phaseextraction（ATPE） Specialcasedofliquid-liquidextractionTwoty
26、pesofaqueoustwo-phasesystems:Polymer-polymertwo-phasesystemle.g.:dextranandPEGPolymer-salttwo-phasesystemle.g.PEGandKClPEGdextransystemTheupperphaseisformedbythemorehydrophobicpolyethyleneglycol(PEG),whichisoflowerdensitythanthelowerphase,consistingofthemorehydrophilicanddenserdextransolution. Aqueo
27、us two-phase extractionBiphasicsystemMonophasicsystemDextran%w/wTielinesPEG%w/w Foreverysubstance,thereisacriticaltemperature(Tc)andpressure(Pc)abovewhichnoappliedpressurecanforcethesubstanceintoitsliquidphase.IfthetemperatureandpressureofasubstancearebothhigherthantheTcandPcforthatsubstance,thesubs
28、tanceisdefinedasasupercriticalfluid.WhatisaSupercriticalFluid?2.SupercriticalFluidExtraction（SFE）Supercritical Fluid ExtractionlSFcombinesdesirablepropertiesofgasesandliquidsSolubilityofliquidsPenetrationpowerofgasesSolvent(SF)SolutePhaseDensity(g/ml)Diffusioncoefficient(cm2/s)Viscosity(g/cm/s)Gas10
29、-310-110-4SCF0.30.910-310-410-410-3Liquid110-510-2The Choice of the SFE SolventCarbondioxideisthemostcommonlyusedSCF,dueprimarilytoitslowcriticalparameters(31.1C,73.8bar),lowcostandnon-toxicity. lDensityofSFandsolubilityofasoluteinitcanbechangedinacontinuousmannerbychangeofpressureSupercriticalFluid
30、ExtractionProcess-flexibilitySFE: TypeslLiquid-SFextractionSimilartoliquid-liquidextractionExamples:lRemovalofalcoholfrombeerlSolid-SFextractionSimilartosolid-liquidextraction(leaching)Examples:lRemovalofcaffeinefromcoffeebeans3.ReversedmicelleextractionlReversedMicelles:surfaceactiveagentorganicsol
31、vent Q: Whats the different between reversed micelle extraction and solvent extraction ? Filtrationa.Proteinsolutionthroughamembranewhichretainstheproteinofinterest.b.Thismethodislesslikelytocausedenaturation.membraneseparationDiffusionmembraneMicrofiltrationUltrafiltrationReverseosmosispressurememb
32、raneElectrodialysis 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.Dialy
33、sisDialysis Typically,processinvolvesseveralchangesofbuffersothatthesaltconcentrationinthesampleisreducedtoacceptablelevel.Whathappensduringdialysis?Whyisdialysisanimportanttechniqueinproteinpurification?Q:Whyisbloodred?Howtotestify?2.Pressuremembrane（Pressuredifferencedrivenprocess）a.Microfiltratio
34、n（MF）Microfiltrationisafiltrationprocesswhichremovescontaminantsfromafluid(liquid&gas)bypassagethroughamicroporousmembrane(0.1to10m).Someexamplesformicrofiltrationb. Ultrafiltration（UF）Usuallyusedtofurtherseparateanycontaminantsabletopassthroughthemicrofiltrationmembraneusingapressuregradient.Smallm
35、oleculesarefilteredoutbypressureUsedforconcentratingproteinsAlternatively,centrifugationwithdialysismembrane超滤浓缩装置超滤浓缩装置 c. c. Reverseosmosis（RO） Osmosisisthe“movementofasolventthroughasemi-permeablemembraneintoasolutionofhighersoluteconcentration”.Theory of OsmosisFreshWaterSeaWaterH2OInitialCondit
36、ionFreshWaterSeaWater(diluted)H2OEquilibriumH2OSemi-permeableMembraneFreshWaterSeaWaterH2OPressureReverseOsmosisRO Membrane Filter DetailIndustrial Applications of RO SystemslPurification of potable or “fresh” water sourceslDesalination of sea water diameterParticlesRecoveryPressureApplicationRFRF 2
37、m 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 v
38、irus 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（electricalpotentialdifferencedrivenpro
39、cess）Electro dialysis（ED）ElectroDialysis(ED)isamembraneprocess,duringwhichionsaretransportedthroughcation-oranion-selectivemembranes,undertheinfluenceofanelectricpotential.SomeExamplesRecoveryofmicroorganismfromfermentationbroth.Removingpyrogens(热原热原)from injection.ConcentrationofmilkExtractingeffectiveingredientsfromdomesticChineseherbs.结束结束
