生物分离工程双语版3

Chapter3

1stHighresolutionfractionationprocessesIIntroduction

(1)appropriatesequence(2)utilization(therapeutic,diagnostic)(3)operationscale(4)roleoffinalfractionationAimpuritiesBsaltsCpathogensDendotoxinsEcontaminatingproteins(5)lowvolumeandexpensiveprocedures(6)specialattentionAamountofproductdenaturationBqualityIIChromatographyprocedures

AgelfiltrationchromatographyBHydroxylapatitechromatographyCHydrophobicinteractionchromatographyDAffinitychromatographyEIon-ExchangeChromatographyAgelfiltrationchromatography(1)mechanismAmolecularsizeBmobilesolventphaseCstationaryphase(2)Rigidityofbeadsinlargescaleoperation(3)traditionalgelfiltrationchromatographymediaASephadexBSuperoseCSephacrylDTrisacrylEBiogelPFCellulofineGFractogel(4)lowproductvolumesuitableforgelfiltration(5)activitylossAfinalfractionationBpreviousbioseparationstepsExmaple3.1carboxylesterasepurification

(1)Material：Bacillusstearothermophilus噬热脂杆菌(2)SteputilizedAcentrifugationBDEAE-Sephacelion-exchangechromatographyCgelfiltrationchromatography(3)ResultanalysisAsinglelow-mobilityband(PAGE)BTwobandsofhighermobility(afterprecipitationandanionexchange）

Cjustsinglebandofhighmobility(furthergelfiltration)DgelfiltrationandSDSindicateesteraseismonomericproteinwithMW40000Exmaple3.1carboxylesterasepurification

(4)ReasonAactivemonomerassociatingmultipleformsBmultipleformhavedifferentchromatographyandelectrophoreticpropertiesCreactionequilibriumaffectedbypH,saltconcentration,enzymedilutionDmonomersormultimericenzymeformsaffectactivityandstabilityDifferencebetweencrudeandpurifiedesteraseforms(1)Thiol-containingcompoundsincrudematerialleadtocysteine-containingproteindenaturation(2)Proteolysisactivityincrudematerial(3)Coprecipitationwithlessstablecompoundsincrudematerial(4)ConformationalchangesinprocessingstepsResultsanddiscussionabouttable3.1(1)48%recoveryishighyield(notsohighpurificationfactor)(2)Statechangeofenzymeaggregationleadtoactivityloss(3)AnalyzecausepermitminimizeactivitylossPurificationofantigenizedimmunoglobulinswithmonomethoxypolyethyleneglycol(1)polyethyleneglycol(PEG)usageAnontoxicBnonimmunogenicCinternal（内用）useinhumansDPEGylatedproteinspreservebiologicalactivity(2)variousdegreeofderivatizationoccurAproteinmicroheterogeneityBdistributionofnumberandportionofPEGCPEGpolydispersity(3)ChemicalreactionmPEGattachedtoAIgsPurificationofantigenizedimmunoglobulinswithmonomethoxypolyethyleneglycol(4)PurificationresultofAIg-mPEGusingsizeexclusionchromatographyAammoniumhydrogencarbonateasbuffersystemBUltrogelAcA-44gelcolumnCtwoelutionpeaksincludingAIgs-mPEGsandunconjugatedAIgs,andmPEG(5)FurtherfractionatingfirstpeakusingQ300anion-exchangecolumnAfirstpeakwithhighPEGylatedIg-Hemaglutinin(HA)BsecondpeakcontainingmildlyPEGylatedIg-hemaglutininCthirdpeakunconjugatedcontrolIg-HA(6)6-8%degreeofderivatizationandlonghalflife

Example3.2Tissueplasminogen

activatorpurification

(1)material：animalcellandbacterial(2)tPA2200USD/dose20timesthanstreptokinase(3)streptokinaseuseonlyonce,tPAmorethanonce(4)tPAmodelproductAflagshipproductforbiotechnolotyBfirstproductformarketusinggeneticalllyengineeredmammaliancellsinsteadofrecombinantbacteria(5)marketrequirementwith11000gtPA/YeartPApurificationprotocols(1)Affinitychromatography(2)Ion-exchangechromatography(3)Aminoacid–Sepharose(4)Gelchromatography(5)Ultrafiltration(6)Centrifugation(6)Microfiltration(7)Solubilization(8)Cleavage(9)refoldingt-PAfromCHOandE.coli(1)5stepspurificationforCHOand16stepsE.coli.(2)Affinitychromatographyandgelchromatographyusedintheendofpurificationprocesstoremoveimpuritiesandsalts(3)t-PApuritygreaterthan99.5%(4)Clearanceofendotoxinwith99.999%(5)Dropinproductyield(refolding20%,ultrafiltration56%Xylanasepurification(1)material:thermophilicascomycete(子囊菌)(2)xylans(majorhemicelluloseinangiosperms)(3)threeseparationstepsAultrafiltrationstepBanionexchangestep(Q-sepharosecolumn)Cgelfiltration(superose12column)(4)xylanaseinteractwithgelfiltrationtoresultinconformationalchange(hightyrosinecontent)PurificationoftwoisoenzymesofxylanasefromstreptomycesPurificationsequence(1)ammoniumsulfate(2)precipitation(3)centrifugation(4)desalting(SephadexG25)(5)DEAE-SepharoseFFcolumn(6)CM-Sepharosecolumn(7)concentrationbyultrafiltration(8)gelfiltrationonSephadexG75column(9)freeze-dryPurificationofhighlythermostableglucoseisomerase(1)material:thermohilicbacteriumThermotogamaritima（2）55%fructoseproducedat95-100C（3）purificationstepAcentrifugationBQ-SepharosecolumnCPhenyl-650MhydrophobicresincolumnDQ-SepharoseHPanionexchangeEHiloadsuperdex200gelfiltrationchromatography(4)SDSASinglebandBWithmolecularweight45000CHomotetramericstructureDMostcommonoligomericstateofglycoseisomeraseExample3.3purificationoftwoendo-β-glucanasefromaerobicfungus(1)material:aerobicfunguspenicilliumcapslatum(2)glycans(聚糖）problemsinbeerindustryAprecipitateBhazes（混浊）

CgelinstoredbeerDgumminess(3)PurificationstepAfreeze-dryofcrudeextractBgelfiltrationonaSephacrylS-300columnCionexchangeonaDE-52celluloseDultrafiltrationEelectrophoresis(4)endo-β-glucanaseAandendo-β-glucanaseB(5)gelfiltrationusedearlier(6)crudeextractismorestableandthepurifiedenzyme.Purificationprotocolforβ-glucosidasefromclostridiumthermocellum(1)material:clostridium(梭菌)therocellum(2)degradationofcrystallinecelluloseproducecellobiosebycellulasecomplex(3)β-glucosidasemetabolizecellobiose(4)purificationstepsAsonicationBcentrifugationCDEAE-SepharoseCL-6BchromatographyDionexchangechromatographyEhighperformanceNonoQHRcolumnFultrafiltrationGchromatofocusingonMonoPHRcolumnHgelfiltration(5)homogeneousenzymeobtainedPectinmethylesterasepurification(1)material:bacillussubtilis(2)pectinismajorstructuralcomponentinplantcellwalls(3)pectinasedegradepectin(4)PMEusageAclarificationofciderBdeesterificationofpectintomethanolandpectate(5)purificationstepsAcentrifugationBultrafiltrationCtwogelfiltration(6)homogeneousPMEobtained(7)proteinlossbyleakageinUFmembrane(PME36000Da,molecularcutoff10000Da)(8)lowactivityinabsenceofsalts(nonspecificbindingoraggregationHydroxyapatitechromatography羟基磷辚石(,Ca10(PO4)6(OH)2)（1）纯化蛋白质的无机介质。（2）廉价，易于大规模应用，（3）使用后易于清洁吸附机理与规律：偶极离子作用而不是离子交换作用(1)每一个正电荷区周围都有负电荷区，反之亦然.

（2）蛋白质在中性pH范围具有最大的形成毗邻正负离子的可能性。工艺条件吸附：(1)pH在6—9之间（2）低浓度缓冲液离子（通常是磷酸盐）（3）低盐浓度洗脱：（1）增加缓冲液浓度（2）高盐浓度中进行（3）盐可以采用恒定或梯度形式应用。BHydroxylapatitechromatographyExample3.5purificationofclostridiumthermoceilumβ-glucosidaseBβ-glucosidaseBusageCatalyzehydrolysisofβ-glucosidelinkagesbetweenglucoseandalkyl,aryl,orsaccharidegroupBioseparationprecedures

AammoniumsulfateprecipitationBQ-sepharoseFFcolumnCbutyl-sepharoseDhydroxylapatitePurificationresultAtable3.9Bstabilityincreasedbyadditionofdivalentcationat45-60CChromatographymediaSilicagelAregularshape(sphere)Buniformsize(3-10µm)CuncoatsurfacedissolvedbyalkalinesolutionDalkylgroupstendtobecleavedinacidicsolutionOthermaterialAtitaniaBzirconia硅胶硅胶是由多聚硅酸经分子间脱水而形成的一种多孔性物质（1）化学组成SiO2.XH20，（2）属于无定形结构，基本结构质点为Si一O四面体，相互堆积形成硅胶的骨架。质点间的空间即为硅胶的孔隙。（3）硅胶中的水以羟基的形式和硅原子相连而覆盖于硅胶表面。硅胶的分类（常以孔径大小来分），(1)特细孔硅胶(0.8nm以下)。

(2)细孔硅胶（1.5—2.0nm)(3)中孔硅胶(4.0一5.0nm)(4)粗孔硅胶(10.nm以上)应用：吸附层析剂与分配层析剂优先吸附极性分子及不饱和的碳氢化合物，用于天然产物分离，可用在水溶液中或有机溶剂中SorbitolandsorbitoldehydrogenaseSorbitolapplication:sweetenerandfoodadditiveSorbitoldehydrogenaseapplication(oxidizessorbitoltoD-fructose)Sorbitoldehydrogenasepurification

A(NH4)2SO4precipitationBQ-SepharoseChromatographyCprocion-blueaffinitychromatographyDhydroxylapatitechromatographyEultrafiltrationPurificatonresultAtable3.10BsingleproteinbandbySDSCpurifiedenzymequitestableandstabilityincreasebyadditionofsucrosePurificationofD-xylulokinaseMaterial:yeastpichia(毕赤酵母属)stipitisD-xylose:ThesecondlargestamountofsugarisnaturallyavailableD-xylullokinase（木酮糖激酶）

convertD-xylosetoethanolPurificationprotocol:TwostepshydroxylapatitechromatographyUltrafiltrationPurificationresultAtable3.11BhomogeneitysampleCmajoractivitylossoccurinthefirstadsorptioncolumnDpartiallypurifiedenzymeunstableat4C,butstableat-20CEallkinasereactionrequiredivalentmetalion(Mg2++)NewwordsandvocabulariesChitanase甲壳素酶Confectioning调制部分,调糖膏）Entail使必须，使承担Rigidity刚性Carboxylesterase羧酸酯酶Esterase酯酶Mobility迁移率Status状况Multimeric多聚体的NewwordsandvocabulariesThiol硫醇

cysteine半胱氨酸Underscore划线，强调MonomethoxypolyethyleneglycolMicroheterogeneity微观异种性Polydispersity多分散性Hemaglutinin凝集素Conjugation结合物Halflife半衰期Tissueplasminogenactivator组织纤维蛋白酶原激活剂Streptokinase链激酶NewwordsandvocabulariesBovinegrowthhormone牛生长激素Chinesehamsterovary(CHO)中国鼠卵细胞Cleavage裂解，分开Xylanase木聚糖酶Ascomycete子囊菌属Hemicellulose半纤维素Angiosperm被子植物Lignin木质素Tyrosine酪氨酸Xylanhydrolase木聚糖水解酶Actinomycetes放线菌NewwordsandvocabulariesSweetener甜味剂Fructose果糖Highfructosecornsyrup高果糖浆Hemotetrameric均一的四聚体Haze混浊Gumminess树胶，粘性Pectin胶质Pectinase胶质酶Texture结构，质地Cider苹果酒Flagellin鞭毛蛋白Hydroxylapatite羟基磷灰石Questions1Whatismechanismofgelfiltration?2Whichdifferencesbetweencrudeproteinsandpurifiedproteins3WhysomeenzymesweremodifiedwithmPEG?4Whystreptokinaseusedonlyonce,whiletPAusedmorethanonce?5Whatismechanismofhydroxylapatite?6Keywords:polydispersity,hemaglutinin,ChineseHamsterovary(CHO),highfructosecornsyrup,hydroxylapatite.

Chapter3

2stHydrophobicchromatographyExample3.7purificationofferuloy/p-coumaroylesteraseMaterial:funguspenicilliumpinophiliumXylans:majorconstituentsofwoodsandagriculturalresiduesP-coumaricesterase:esterifiestheL-arabinoseresiduesinthexylanbackbone(importantmodificationPurificationprocedures:AAmiconultrafiltrationBDEAE-SepharoseCL-6Banion-exchangechromatographyChydrophobicinteractioncolumnPurificationresultAtable3.12BSinglebandbySDSand57000DatonMWCstabilefrom37-55C疏水层析

将疏水性基团如丁烷、辛烷、苯固定化到介质上,这些基团会与蛋白质生物大分子上的疏水区亲和介质制备：琼脂糖在有机溶剂中用CDI(羰基二咪唑)活化后再与芳胺或烷胺形成酰胺键得到疏水层析介质介质配基密度:40—80μmol/ml胶,吸附容量：牛血清蛋白(BSA)大于40mg/ml疏水层析介质制备过程示意图工艺条件吸附：(1)盐浓度：高盐，1—2M(NH4)2SO4

或NaCl(2)pH:中性或偏酸性淋洗：0.5—2%表面活性剂洗脱:(1)低盐，0.1—0.5MNaCl,再配合pH变化。(2)低浓度促溶剂,0.1—0.5%NaSCN,或20—40%乙二醇疏水层析实验方案：NewwordsandvocabulariesHydroxylapatite羟基磷灰石Alkyl烷基Aryl芳基Saccharide糖类Butyl-Shpharose丁基-SepharoseSilicagel硅胶Uncoated未涂层Titania二氧化钛Zirconia氧化锆Sorbitol山梨醇D-glucitol葡萄糖醇Acyclicpolyol无环多醇Nicotinamideadeninedinucleotide(NAD)尼克酰胺腺嘌呤二核苷酸Xylitol木糖醇Sucrose蔗糖D-xylulokinase木酮糖激酶Pentose戊糖Phosphorylation磷酸化Feruloyl/p-coumaroylesterase（氯苯二酚氨醇香豆素脂酶）Arabinose树胶醛糖，阿拉伯糖AffinitychromatographyExample3.8purificationofchitanaseMaterial:trichoderma（木霉）barzianumPurificationprotocolAammoniumsulfateprecipitationBQ-Sepharoseion-exchangechromatographyCSephadexG-100gelfiltrationDphenyl-SepharoseCL-4BhydrophobicinteractionchromatographyPurificationresulttable3.13ClustermodelofmultivalentaffinitySelectivityisdependentonligandandmatrixMultivalentinteractionAtwoormoreligandinaclusterbindingtwoormorecontactsBcooperativityduetoproximityandentropicCassociationconstantsforligandpairs100-foldgreaterthanconstantforsingleligand,whilemodelconstantshouldbesevenordersofmagnitudehigherDgeometrical-stericconstraint

亲和层析亲和层析(AffinityChromatography)是利用生物体内存在的特异性相互作用的分子对而设计的层析方法。生物体内相互作用的分子对：(1)酶—底物或抑制剂(2)抗原—抗体。(3)激素—受体。(4)糖蛋白与凝集素,(5)生物素—生物素结合蛋白等

基质活化方法与配基偶联

活化(activation):

基质（matrix）上的化学基团是不活泼的,无法与配基直接偶联,通过化学反应使介质上化学基团处于活化状态。(1)大分子配基如蛋白质等可与活化基质直接偶联。(2)小分子配基,

在基质与配基之间插入若干碳原子手臂(spacer),然后再与配基偶联。(3)环氧氯丙烷,1,4－丁二醇缩水甘油醚本身是活化剂亦具有手臂作用。活化方法（1）A溴化氰法溴化氰在碱性条件下与多糖上的羟基反应导入氰酯键或亚氨碳酸酯到基质上,进而与配基偶联优点:（1）适用于含羟基多糖及含羟基的合成基质（2）用于含伯氨基小分子配基及伯氨基大分子配基的偶联（3）操作步骤简单,重现性好。（4）偶联条件温和,特别适用于偶联敏感性生物大分子。缺点：（1）形成的异脲键易产生非特异性吸附,（2）共价键不稳定,配基容易脱落,（3）活化操作危险性大，反应后的残余液需经处理再排放

溴化氰活化与配基偶联化学反应式

活化方法（2）B环氧氯丙烷活化

（1）所形成的共价键稳定,配基不易落脱（2）自动引入手臂,（3）有更小的非特异性吸附,（4）活化操作简单易行,危险性相对较小缺点：在强碱性条件反应，不适用于碱敏感物质。环氧氯丙烷活化反应式

环氧基的氨化及偶联配基反应Ion-exchangechromatographyExample3.10purificationofk-carrageenaseMaterial:pseudomonas（假单孢菌属）carrageenovoraCarrageenans（角叉菜聚糖）:majorcomponentofcellwallstructureinredalgaeCellwalldegradingenzyme:usedforobtainingprotoplastsandunderstandingstructureofcarrageenansPurificationprotocolSmallscaleA(NH4)2SO4precipitationBSephadexcolumngelfiltrationCCM-sepharoseCL-6BcolumnLargescaleAultrafiltrationBS-SepharoseFFcationexchangePurificationresult

Atable3.14Bdoubleband(32000and34000Da)离子交换法概述：利用离子交换树脂分离生物物质的方法特点：（1)浓缩效应

(2)成本低，设备简单，操作方便

(3)生产同期长，pH变化大离子交换树脂：不溶于酸碱和有机溶剂的固态高分子化合物骨架活性离子强酸性阳离子交换树脂-SO3H，-PO（OH）2-PHO（OH）RSO3H+NaClRSO3Na+HCl交换能力与pH无关弱酸性阳树脂-COOH，-OH(酚羟基）

强碱性阴树脂

弱碱性阴树脂Example3.11Productionofbloodprotein

usingion-exchange

Material:humanplacentalbloodandhumanplasmaMatrix:silicaparticlesArigidityBincompressibilityClowpressuredropDlargeporediameterEcoatedbysuitablepolymerssuchaspolysaccharideFpossibleapplicationonanindustrialscaleResultsanddiscussion

AProductionscale20000liters/dayand140KgofproteinBSucroseandalbuminminimizedenaturationofbloodcoagulatingfactorCExtensivedialysisordiafiltrationleadtodenaturationofproteinswhenremovalofcompetitiveligandsDMildelutionconditionswithmetalchelatingagentsminimizemetalleakage离子交换提取蛋白质传统离子交换剂不适用于提取蛋白质（1）交联度大（大分子不能进入）（2）电荷密度高(结合太强）（3）骨架憎水性强（蛋白质易变性)亲水性离子交换剂（1）亲水性大（2）孔径大（3）体积随pH及离子强度变化小亲水性离子交换剂功能团离子交换剂的交换容量亲水性离子交换容量不能达到无机离子交换容量（1）蛋白质不能进入活性中心（2）一个蛋白质与多个功能基发生作用吸附机理

（1）静电吸力（2）憎水（3）氢键（4）被吸附蛋白表面进一步吸附蛋白质的离子交换层析层析介质：DEAE-纤维素，CM-纤维素吸附条件：

(1)缓冲液pH(2)蛋白浓度控制（5mg/ml)（3）离子强度

(4)道南效应(Donnaneffect)介质内外盐浓度及

pH不同

(5)上样量：1-5%BV，高径比20:1,L：100cm(恒定洗胶）；5-10%qm,柱长度20-40cm,高径比5：1

（分步或梯度）洗脱：恒定，分步，梯度pH梯度：a缓冲量大；b离子强度变化；cpH变化大NewwordsandvocabulariesAcetamido乙酰氨基Endoglycosidase葡萄糖内酯酶Phenyl-SepharoseCL4B苯基-P-nitrophenyl-β-N-actylglucosamine对硝基苯-N-乙酰葡萄糖胺Entropic熵Rabbitmuscledehydrogenase兔肌脱氢酶Clusters簇Lactatedehydrogenase乳酸脱氢酶CibacronbluecelluloseOrdersofmagnitude数量级NewwordsandvocabulariesRandom随机K-carrageenasek-角叉菜酶

algae海藻Seaweed海草，海藻Protoplasts原生质体CM-SepharoseCL-6BPhycocolloid藻胶Placental胎盘的Chloroform氯仿Hemoglobin血红素Electrolytes电解质IIICrystallizationCrystallizationofbacterialluciferase（萤光素酶）Material:marinebioluminescent（生物发光）

bacteriumCrystallizationsteps(1)microfiltration(2)sitting-dropvapordiffusionmethodwithcrystallizationplates(3)Biomek1000automatedlaboratorysystem(accuracy,reproducibility,andprecision)Example3.12purificationandcrystallizationoflipaseMaterial:geotrichum（地丝霉属）candidum（念珠菌）Lipasehaveahighspecificityforfattyacidshavingatleastonecis-Δ9doublebond.Purificationsteps(1)Q-Sepharosecolumn(2)Phenyl-SepharosecolumnCrystallization

(1)hanging-dropmethodusingmultichamberplates(2)crystallizedinthepresenceofPEG(3)crystalssizedependsonmolecularweightofcrystallizationagent(4)fivedifferentcrystalsobtaineddependingontheamountofglycosylation(5)pureenoughtoobtainX-raydiffractiondataPurificationandcrystallizationoflipasePurificationProtocol(1)ion-exchangechromatography(2)S-Sepharosefastflow(3)Phenyl-SepharoseCL-4Bhydrophobicinteractionchromatography(4)hydroxylapatiteadsorptionchromatography(5)Ω-aminohexylagaroseaffinitychromatographyCrystallization

(1)bundlesofneedlesobtainedinammoniumsulfite(2)rhombic(斜方）crystalsobtainedinammoniumsulfate(3)purecrystalweresuitableforX-raydiffractionstudiesAntibioticscrystallizationExample3.14penicillincrystallizationBasicbioseparationprotocol(1)filtration(2)centrifugation(3)liquid-liquidextraction(4)crystallizationDiscussion(1)purificationofantibioticsundersterilecondition(2)rapidprocessing(3)sensitivetotemperature(4)extracaretopreventcontaminationordegradation(β-lactamase-producingorganisms)Example3.15purificationandcrystallizationofcephalosporinAntibioticscrystallizationBioseparationprotocol(1)solventextraction(2)ion-exchangeresin(3)salting-out(4)activatedcarboncolumn(5)precipitation(6)crystallizationApotassiumorsodiumsaltofcephalosporinBmisciblesolventCCoper,nickel,orleadsaltcrystallizationfromaqueousssolutionsIVothertechniquesExample3.16purificationofβ-galactosidaseMaterial:Aspergillus（曲霉）fonsecaeusβ-galactosidase:hydrolysisoflactoseinmilkandwheyindairyindustryProtocol(1)ultrafiltraction-diafiltration(2)isopropanolprecipitationPurificationresult(1)table3.18(2)isopropanolprecipitationashigh-resolutionfractionationstep(3)diafiltrationtoremovemajorlowmolecular-weightcontaminants(4)furtherpurificationAhydroxylapatiteBgelfiltrationCanionexchange(Fastphaseliquidchromatography,FPLC)(5)bothpartialpurificationandhighpurificationprovidedExample3.17purificationofβ-glucosidaseMaterial:fungusNeocallimastixfrontalisEB188(1)Protocol(2)ConcentrationbyMiliporefilter(3)Ethanolprecipitation(4)Hydroxylapatitechromatography(5)Gelfiltrationchromatography(6)Ion-exchangechromatography(7)nativePAGEResultsandDiscussTable3.18HydroxylapatitechromatographytwiceAfirsttreatmentobtainedfourpeaksBsecondtreatmentobtainedtwopeaksExample3.18separationofperoxidaseMaterial:soybeanhulls（壳）