酶的分離工程-1課件

Chapter4

PurificationofEnzymesProteinsareverydiverse.Theydifferbysize,shape,charge,hydrophobicity,andtheiraffinityforothermolecules.Allthesepropertiescanbeexploitedtoseparatethemfromoneanothersothattheycanbestudiedindividually.Objectives

PurityStableCostTimePurificationtypicallyinvolvesthreesteps1)Preparationofacrudeextractfromharvestedcells2)Fractionation:Separationofamixtureofproteinsintovariousfractionsaccordingtosomeproperty(e.g.size,charge,solubility)3)SeparationofproteinfromsolventsandconcentrationUnit1PreparationofCrudeEnzymes

Endoenzyme:intracellularMostenzymesofthemetabolicpathways.Exoenzyme:extracellularBreakdown(hydrolyze)largefoodmoleculesorharmfulchemicals.Example:cellulase,amylase,penicillinase.

Solid/LiquidSeparation

-----centrifugation

and

filtrationWhenharvestingbrothcultures,howarecellsseparatedfromthebroth?DecanterCentrifugeClarifiedliquidRotatingBowlRotatingscrollHowtoimprovefiltervelocity?

1）FlocculationandAgglomeration

2）

Decreaseviscosity

3）Filteraid

CellDisruptionThemaincomponentofcellwall

Bacteria

:

Peptidoglycan

Yeast：Dextran，Mannose，Protein

Mycelialfungus:Chitin,Dextran

GramPositiveBacterialCellWallFungusCellWallMechanicalmethodsGrinding(inliquidnitrogen,ballmill）－DrywayHomogenization（mortar,homogenizer)-WetwayPhysicalmethodstemperaturedifference（

freezingandthawing）pressuredifference(

osmoticshock)ultrasonicationChemicaltreatmentorganicsolventsdetergents：TritonX-100，Tween

（usedifenzymeisinlipidmembrane）Enzymelysisautolysis

extraenzymeWaystobreakcell

BeadMillCascadingbeadsCellsbeingdisruptedRollingbeadsAfterbreakingthecell……1)Keeptemperaturelow2)Purifyassoonaspossible3)Avoidoxidation4)AvoidcontaminationCoolingandproteaseinhibitionareimportanttorecovertheenzyme!

EnzymeExtraction

Fromplantandanimaltissue.

Toachievemaximumsolubilityandactivityoftheenzyme.

ExtractmethodsSolventorSolutionextracttargetsaltingliquid0.02～0.5mol/LNaClsolutionacidsolutionpH2～6aqueous

solutionalkalisolutionpH8～12aqueous

solutionorganicsolventwater-miscibleorganicsolventMethodsforExtractionofEnzymes

Unit2MethodsofPurification

Centrifugation

Preparativecentrifugation

Analyticalcentrifugation

Preparativecentrifugation?Collectmaterialcellsprecipitatedmacromolecules?SubcellularfractionationAnalyticalCentrifugation

SedimentationCoefficient(s)?isthevelocityperFc,ors=v/ω2r?unitisSvedberg,where1S=10-13secSpeed(rpm)

Importantnoticelowspeedcentrifuge6000R.T.equilibriumhighspeedcentrifuge6000?25000freezingequilibriumexactlyultraspeedcentrifuge

>25000freezing＋vacuumsystemequilibriumexactlyTypesofCentrifuges1）DifferentialCentrifugation

(GravityCentrifugation)

Separatesupernatantandpelletbymassanddensity?preparecelllysate?subjecttocentrifugation

centrifugalforcetime(g·min)tubesizeandshaperotorangle?re-centrifugesupernatantProblems?contamination

largeparticlescontaminatedwithsmallerparticles?resolutionparticlesofsimilarsizesnotseparated?vibrationsandconvectioncurrents2)SedimentationVelocityRateZonal≡ρp>ρs(大)?separatesprimarilybymasscommonmedia:sucrose3)SedimentationequilibriumIsodensity≡ρs（?。?/p>

<ρp<ρs（大）?equilibrium?separatesbydensitycommonmedia:CsCl

每種純樣品成份在梯度液中的沉降速度（cm/s）

d2(ρp

-

ρs)ω2rV=18η式中d：樣品顆粒的直徑（cm）

ρp

：樣品顆粒的密度（g/cm3）

ρs：密度梯度液的密度（g/cm3）

當ρp>ρs時,

V>0,樣品順離心力方向沉降

ρp<ρs時,

V<0,樣品逆離心力方向上浮

ρp

=

ρs時V=0,樣品停止沉降或上浮，"穩(wěn)定"在這一位置CommonFeaturesCentrifugationinadensemedium-increasesstability-providesgreaterresolution

ComparisonofcentrifugesGravityCentrifugation

(Nodensity)UtilizegravityforcetoseparateparticlesfromthesolutionDifferentialCent.

DensityGradientCent.ZoneCentrifugation

(Precast)(step-wise)Isodensityequilibration(CsClgradientforming)Sample:protein(similardensity,butdifferentinMW)Sample:nucleicacid(similarMW,butdifferentindensity)HighspeedUltracentrifugationTwoultracentrifugetypesCentrifugeSedimentationvelocitySedimentationequilibriumAlsocalledZoneCentrifugationIsodensityequilibrationGradientformationPrecast(sucrose,glycerol)Shallowgradient,lowerdensityDuringcentrifugation(CsCl)Steepgradient,higherdensitySuitablesamplesSimilardensity,differentMWProteinSimilarMW,differentdensityNucleicacid/cellorganelleCentrifugationconditionsLowerspeed,notcompletesedimentated,stopatpropertimeCompletelysedimenttowherethedensityisequilibrated,highspeed,longrunningtimeGeneralProceduresPreparegradient2)Applysample3)Centrifuge4)CollectandanalyzefractionsLarge-scalePurificationofViruses1.

Growthofthevirusinlarge-scaleequipment2.Removalofvirus-enrichedculturefluid3.Concentrationofthevirusparticlesbyprecipitation4.Finalpurificationbydensity-gradientcentrifugation5.CrystallizationofvirusPrecipitation——Decreasesolubility

Thisunitoperationservestoconcentrateandfractionatethetargetproductfromvariouscontaminants.1.Saltingout

——Changeinionicstrength

Salteffectsproteinsolubility------Saltingin

:

Additionofsaltatlowionicstrengthcanincreasesolubilityofaprotein.Saltingout:Proteinstendtoaggregateandprecipitatefromhighionicstrengthsolution.Saltingin

ProteinhasnonetchargeatitspI,thatleadstothebindingbetweenproteinsviaionicinteractions,andprecipitation.Saltcaninterferetheseionicinteractionsandseparateboundproteinmolecules.Salting-ineffect:decreaseprotein－proteinelectrostaticinteraction;solubilityincrease!

Saltingout

(Canbeusedforfractionation)

Beyondacertainionicstrength（>0.1M）,thechargedmoleculesarequicklyprecipitatedbecausetheexcessions(notboundtotheprotein)competewithproteinsforthesolvent.

Salting-outeffect:ionstake‘a(chǎn)ll’water,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（飽和度）

Volumeofsaturated(NH4)2SO4Saturationratio＝

TotalsolutionvolumeHowtoachievedesiredpercentageofammoniumsulfate？

Add……

-saturatedsolution-drypowderHowtomakingX%solutionfromXo%solution?Theeffectofsaltondifferentproteinsmaydiffer:Certainproteinsprecipitatefromsolutionunderconditionsinwhichothersremainquitesoluble.Oncetheproteinisprecipitated(notdenatured)–canseparatebycentrifugationpelletcanberedissolvedinbufferforfurtherpurification

?Whichproteinwillpptfirst?(hydrophobicorhydrophilic?)Fractionalsaltingout——Differentproteinsprecipitateatdifferentsaltconcentration.serumglobulinalbumin(NH4)2SO450%saturationsaturatedprecipitateprecipitate

Saltingoutcurveprotein(mg)orenzymeactivity

102030405060708090100(NH4)2SO4

percentageofsaturated

Inbrief,theproceduregoesasfollows:obtainproteinsolutionofinterest2)add(NH4)2SO4toachilled,stirringsolution3)allowtostirfor15-30minutes4)collectprecipitatedproteinbycentrifugation5)

re-dissolvedinbufferforfurtherpurificationImportantfactors:1)ionicstrength

S=solubilityoftheproteinKs：salt-specificconstantβ:idealizedsolubilityI:theionicstrengthofthesolutionlogS=β-KsI2)pH:≈pI3)temperaturelowionicstrength,T.

proteinsolubilityhighionicstrength,T.

proteinsolubility4)proteinconcentration:moderate2.Precipitationwithorganicsolvents——Decreaseindielectricconstant

Organicsolventdecreasesthewateractivityandthedielectricconstantofthesolution,whichthendecreasesthesolubilityoftheproteinandprecipitatesit.Commonorganicsolvent:

acetoneethanolmethanol，≈2×proteinvolumeImportantfactors1)Temperature：low（0℃）

Because......a.Someproteinsmightbedenaturedbyheatproduced.b.Increaseenzymeyield（T.

,solubility）2)pH：≈pI3)

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.Comparisonoftwomethods

3.Isoelectricpointprecipitation

——ChangeinpH-

EnzymesareleastsolubleatpI-DifferentenzymeshavedifferentpI

UsingmethodItseldombeusedalone（oftenusedtoremoveundesiredprotein）.Because：ManyproteinshavesimilarpI.ProteinshavesomesolubilityatpI.

4.Non-ionichydrophilicpolymers

（Polyethyleneglycol（PEG）precipitation）Molecularweight:6000～20000RemarksPEGwillprecipitateswithoutdenaturing.Itsprecipitationeffectsisveryhigh.

5.SelectivedenaturationAnegativemethod——leavesthedesiredproteinactiveinsolution;

heat;

extremepH;

organicsolvents;Relationshipbetweendenaturationandprecipitation?Casein(酪蛋白),denaturedinboilingmilk,willnotbeprecipitated.Proteinisnotdenaturedbysaltingout.Extraction

Amethodtoseparatecompoundsbasedontheirrelativesolubilitiesintwodifferentimmiscibleliquids.

Organicsolventwater

1.Aqueoustwo-phaseextraction（ATPE）

Specialcasedofliquid-liquidextractionTwotypesofaqueoustwo-phasesystems:Polymer-polymertwo-phasesystem e.g.:dextranandPEGPolymer-salttwo-phasesystem e.g.PEGandKClPEG–dextransystem

The"upperphase"isformedbythemorehydrophobic

polyethyleneglycol(PEG),whichisoflowerdensitythanthe"lowerphase,"consistingofthemorehydrophilicanddenserdextransolution.Aqueoustwo-phaseextraction

BiphasicsystemMonophasicsystemDextran%w/wTielinesPEG%w/w

Foreverysubstance,thereisacriticaltemperature(Tc)andpressure(Pc)abovewhichnoappliedpressurecanforcethesubstanceintoitsliquidphase.IfthetemperatureandpressureofasubstancearebothhigherthantheTcandPcforthatsubstance,thesubstanceisdefinedasasupercriticalfluid.WhatisaSupercriticalFluid?2.SupercriticalFluidExtraction（SFE）SupercriticalFluidExtractionSFcombinesdesirablepropertiesofgasesandliquidsSolubilityofliquidsPenetrationpowerofgasesSolvent(SF)SolutePhaseDensity(g/ml)Diffusioncoefficient(cm2/s)Viscosity(g/cm/s)Gas10-310-110-4SCF0.3～0.910-3～10-410-4～10-3Liquid110-510-2TheChoiceoftheSFESolventCarbondioxideisthemostcommonlyusedSCF,dueprimarilytoitslowcriticalparameters(31.1°C,73.8bar),lowcostandnon-toxicity.

DensityofSFandsolubilityofasoluteinitcanbechangedinacontinuousmannerbychangeofpressureSupercriticalFluidExtractionProcess-flexibilitySFE:TypesLiquid-SFextractionSimilartoliquid-liquidextractionExamples:RemovalofalcoholfrombeerSolid-SFextractionSimilartosolid-liquidextraction(leaching)Examples:Removalofcaffeinefromcoffeebeans3.ReversedmicelleextractionReversedMicelles:

surfaceactiveagent＋organicsolventQ:What’sthedifferentbetweenreversedmicelleextractionandsolventextraction?FiltrationProteinsolutionthroughamembranewhichretainstheproteinofinterest.Thismethodislesslikelytocausedenaturation.membraneseparationDiffusionmembrane

Microfiltration

Ultrafiltration

ReverseosmosispressuremembraneElectrodialysis

dialysisElectromembrane

1.Diffusionmembrane（concentrationdifferencedrivenprocess）

——dialysisDialysistubinghasporeswithaspecificmolecularweightcut-offthatallowssmallermolecules(salt)topass.Purposes:Reduceionicstrengthofthesolution.Concentrateproteinsample.Dialysis

Typically,processinvolvesseveralchangesofbuffersothatthesaltconcentrationinthesampleisreducedtoacceptablelevel.Whathappensduringdialysis?Whyisdialysisanimportanttechniqueinproteinpurification?

Q:Whyisbloodred?Howtotestify?2.Pressuremembrane（Pressuredifferencedrivenprocess）Microfiltration（MF）Microfiltrationisafiltrationprocesswhichremovescontaminantsfromafluid(liquid&gas)bypassagethroughamicroporousmembrane(0.1to10μm).Someexamplesformicrofiltrationb.Ultrafiltration（UF

）Usuallyusedtofurtherseparateanycontaminantsabletopassthroughthemicrofiltrationmembran