BioseparationEngineering課程性質(zhì)及建設(shè)歷程集美大學(xué)開設(shè)生物工程專業(yè)2000集美大學(xué)校級(jí)特色專業(yè)福建省服務(wù)產(chǎn)業(yè)特色專業(yè)福建省一流本科課程200820162020福建省一流專業(yè)建設(shè)點(diǎn)20232025國家一流本科課程課程性質(zhì)及建設(shè)歷程教學(xué)理念及過程授課方式授課內(nèi)容學(xué)時(shí)數(shù)線上基礎(chǔ)理論知識(shí)與應(yīng)用10線下22企業(yè)導(dǎo)師專題討論

4英文文獻(xiàn)匯報(bào)4合計(jì)40課程安排及考試方式考試組成組成總成績占比期末考試成績基本知識(shí)（60%）60%閱讀英文文獻(xiàn)（40%）平時(shí)成績線上學(xué)習(xí)（15%）40%文獻(xiàn)匯報(bào)（15%）作業(yè)（10%）考勤規(guī)定：缺席6次及以上不能參加期末考試。每缺課一次平時(shí)成績總分扣3分。References（1）《AdvancesinProteinPurification》，CarolineGardner，CallistoReference，2015.（2）《ProteinPurification:PrinciplesandTrends》，IconceptPress，2016（3）《生物分離與純化技術(shù)》，李從軍,郭麗娜，四川大學(xué)出版社，2018.（4）《生物分離科學(xué)與工程》，余潤蘭，中南大學(xué)出版社，2018.（5）《生物技術(shù)在海洋生物資源開發(fā)中的應(yīng)用》，王梁華,焦炳華，科學(xué)出版社，2017.（6）《生物分離工程》，胡永紅，李鳳珠，韓矅平主編，華中科技大學(xué)出版社，2015.（7）《生物分離工程》，孫彥，化學(xué)工業(yè)出版社，2005（8）《PROTEINEXPRESSIONANDPURIFICATION》，ACADEMICPRESSINCELSEVIERSCIENCE，雜志Textbook《生物分離工程》，李健、陳超翔主編，曹敏杰主審.化學(xué)工業(yè)出版社，2025；福建省“十四五”規(guī)劃教材、化學(xué)工業(yè)出版社“十四五”普通高等教育規(guī)劃教材BiochemicalProductsChapter1introductionChapter2filtration（過濾）andcentrifugation（離心）Chapter3celldisruption（細(xì)胞破碎）Chapter4crystallization（結(jié)晶）andprecipitation（沉淀）Chapter5membraneseparation（膜分離）Chapter6extraction（萃?。〤hapter7chromatography（色譜、層析）Chapter8electrophoresis（電泳）andmagneticbioseparation（磁性生物分離）Chapter9solventremoval（溶劑去除）anddrying（干燥）Chapter1Introduction1.1Introduction1.2ClassificationofbioseparationSteps1.3Bioseparationprocessselection1.4Developmentsofbioseparationprocess基本要求：掌握生物分離工程在生物工程領(lǐng)域的地位，生物分離過程的特點(diǎn)以及生物分離過程的分類。了解生物分離過程選擇原則及發(fā)展。重點(diǎn)：準(zhǔn)確理解生物分離過程的特點(diǎn)；生化分離工程的一般流程和單元操作難點(diǎn)：生物分離過程的特點(diǎn)；生化分離工程的一般流程。

1.1introductionBiochemicalengineeringBioprocessingDownstreambioprocessing(bioseparationengineering)CharacteristicofBioseparationsBiochemicalengineering（生化工程）isconcernedwithconductingbiologicalprocesses

onanindustrialscale.Thisareacombinebiologicalsciences

withchemicalengineering.Bioprocessing（生物過程）:manufactureofbiologicalproducts.IndustrialchemicalsAgrochemicalsPharmaceuticalsFoodandfoodadditivesNutraceuticalsDiagnosticproductsCommoditychemicalsLaboratoryreagentsCosmeticproductsBioprocessingincludesthreeessentialstages:upstreambioprocessing:cellularexpressionmidstreambioprocessing:bioreactordownstreambioprocessing:productpurification

Downstreamprocessingorbioseparationengineering（生物分離工程）:systematicstudyofthescientificandengineeringprinciples

utilizedfortherecovery（提取）,isolation（分離）,purification（純化）andpolishing（精制）ofbiologicalproducts.CharacteristicsofbioseparationBiologicalproductsexistinhighlydilutedaqueoussolutionsinthebioreactor.Mostofbiologicalproductsareunstable.Biologicalproductsrequirehighquality

（safety,identity/purityandpotency/activity）Thecomponentsinthefeedarecomplicate.Manycurrentcommercialproductsaremanufacturedinbatches.尿激酶干擾素紫杉醇生長激素?zé)晒馑孛敢葝u素腎素四環(huán)素青霉素膠原蛋白丁醇乙醇山梨糖醇檸檬酸乳酸生物反應(yīng)器細(xì)胞收集細(xì)胞破碎提取純化產(chǎn)品制劑Bioseparationprocessflowdiagramforintracellularproducts1.2

ClassificationofbioseparationSteps1.2

ClassificationofbioseparationStepssolid-liquidseparationRecoveryPurification

（polish）finalproductformulationexamplessolid-liquidseparationOperationspretreatment（預(yù)處理）filtration（過濾），centrifugation（離心），microfiltration（微濾）,sedimentation（沉降）celldisruption（細(xì)胞破碎）(necessarywhenthedesiredproductisintracellular.)Relativelylittleproductconcentrationorimprovementofproductqualityoccurs.

RecoveryOperationsadsorption（吸附）extraction（萃?。﹑recipitation（沉淀）membraneseparation

（膜分離）Thesesteps,whicharerelativelynonspecific,removematerialsofwidelydivergentpropertiescomparedtothedesiredproduct.（以上分離過程不具備特異性，只是進(jìn)行初分）Appreciableconcentrationandproductqualityincreasesusuallyoccur.（可提高產(chǎn)物濃度和質(zhì)量）purificationOperationschromatography（色譜）electrophoresis（電泳）crystallization（結(jié)晶）Theseprocessingtechnologyarehighlyselective

fortheproductandremoveimpuritiesofsimilarchemicalfunctionalityandphysicalproperties.（以上技術(shù)具有產(chǎn)物的高選擇性和雜質(zhì)的去除性）finalproductformulationOperationsconcentration（濃縮）drying（干燥）sterilize（滅菌）removingpyrogen（去除熱源）addingexcipients（加賦形劑）

thefinalstateinwhichtheyarepackaged,stored,anddispensed.Asuccessfulformulationprocesswillofferbothefficiencyandsafety.Theresultingproductmustremainstable.BioreactorfluidwithcellsContinuouscentrifugation100000MW

membraneAffinityChromatographyUnboundmaterialCellsSterilefiltrationformulationMembraneconcentrationIgGproductIgGelutedSupernatantFigure1.1Recoveryandpurificationofmonoclonalantibodies單克隆抗體MW：160000daltons，分泌胞外蛋白A或蛋白G超濾超濾BioreactorfluidwithcellsfiltrationActivatedcarbontreatmentfiltrationAcetone丙酮andwaterandsodiumorpotassiumacetate

乙酸鈉、乙酸鉀Figure1.2Recoveryandpurificationofpeniclillin青霉素Amylacetate乙酸戊酯ExtractionspH2-3dryingFiltrationFiltrationcrystallizationWashingproductIsopropanol異丙醇o(jì)rbutanol丁醇SpentsolventSpentsolventT=5℃CaCl2andpolyelectrolyte聚合電解質(zhì)filtrateextractSpentsolventCarbonandimpuritiesCentrifugationorfiltrationpH-adjustedAdsorptionbyacidicresinEvaporation

HClHCl帶＋2靜電荷dryingAnimalfeedproductElute洗脫Figure1.3animalandfood-gradel-lysinerecoveryandpurificationAnimalfeedorfood-gradeproductsfiltrationcrystallizationfiltrationdryingFood-gradeproductpH-adjusted陽離子交換樹脂，氯化銨洗脫L－lys－HClActivatedcarbon1.3bioseparationprocessselectionhighthroughput/productivitylowcostusingthefewestnumberofprocessstepssequenceofbioseparationstepsqualityrequirementsandregulatoryenvironmentproductionscalecomponentsoffeedlocationandnatureofproductwasteproductionanddisposalcostCapitalcostDesigning,purchasing,installingapieceofbioseparationequipmentOperatingcostmaintenanceutilitiessuchaselectricity,steamandcompressedairlaborrawmaterialsTherecoveryplantusuallyrepresentsamajorinvestmentand,consequentlytheseparationscostasubstantialfractionofthetotalcostofthefinalproduct.（分離過程的成本占產(chǎn)品總投資的大部分）ProductBioseparationcost(%)Solventse.g.ethanol（乙醇）,acetone（丙酮）15-20Cells,e.g.bakersyeast（面包酵母）,brewersyeast（啤酒酵母）20-25Crudecellularextracts,e.g.yeastextract（酵母提取物）20-25Organicsacids,e.g.citricacid（檸檬酸）,lacticacid（乳酸）30-40Vitaminsandaminoacids

e.g.lysine,ascorbicacid（維生素C）30-40Antibiotics,e.g.penicillins20-60Industrialenzymes,e.g.glucoseisomerase(葡萄糖異構(gòu)酶)40-65Non-recombinanttherapeuticproteins,e.g.pancreatin（胰酶）,papain（木瓜蛋白酶）50-70Monoclonalantibodies50-70Nucleicacidbasedproducts60-80Plasmaproteins,humanalbumin,humanimmunoglobulins70-80usingthefewestnumberofprocessstepsInordertogetahighproductivity,IncreasetherecoveryofeachbioseparationstepReduce

the

number

ofoperationstepsForexample:2steps90%Recovery：90％×90％＝81％3steps90%Recovery：90％×90％×90％＝72.9％natureofproductsolubility

溶解度electrostaticcharge靜電荷

size大小functionalgroup

功能基團(tuán)volatility揮發(fā)性stability穩(wěn)定性hydrophobicity疏水性1.4DevelopmentsofbioseparationprocessImprovetheconventionalseparationtechniques.Developnewtechniques,newmaterialsandnewequipments.CombinationmethodsImprove

upstreambioprocessingandmidstreambioprocesssing,andsimplify

downstreambioprocessing.Questions1.Whatisthedifferencebetweenbiologicalproductsandordinarychemicalproducts?2.Whatfactorsshouldbeconsideredindesigningseparationprocessesforbiologicalproducts?3.Howtocalculatetheyieldofpurifiedbiologicalproducts?Iftheyieldofproductspurifiedineachstepis90%,whatisthetotalyieldin6steps?生物反應(yīng)器細(xì)胞收集細(xì)胞破碎提取純化產(chǎn)品制劑BioreactorExtractionRecoveryCelldisruptionCellharvestingPurificationProductformulationPrecipitationandcrystallizationSupernatantChapter2Filtrationandcentrifugation2.1pretreatment2.2filtration2.3centrifugation基本要求：掌握過濾前物料預(yù)處理的基本方法，了解轉(zhuǎn)鼓過濾器的操作原理與過程，以及過濾設(shè)備的基本結(jié)構(gòu)；了解管式離心機(jī)、碟片式離心機(jī)及螺旋式離心機(jī)，掌握差速離心方法和密度梯度離心方法。2.1pretreatmentCharacteristicsoffermentationbrothPretreatmentmethodsHeatingAdjustingpHCoagulationandflocculation凝聚和絮凝Addingfilteraids助濾劑上的吸附Addingreactant反應(yīng)劑Methodsforremovingimpurities

protein,insolublecarbohydrates,multi-valentmetalionsCharacteristicsoffermentationbrothviscous,highlynon-NewtonianslurryverydiluteaqueoussystemssmallparticlesizeminimaldensitydifferencesbetweenthesolidandtheliquidphasehighsolidscompressibilityManyfermentationbrothsareunstable.發(fā)酵viscous,highlynon-Newtonianslurrysmallparticlesize

minimaldensitydifferencesbetweenthesolidandtheliquidphasesmallparticlesize

minimaldensitydifferencesbetweenthesolidandtheliquidphaseVShighsolidscompressibilityFiltrationcentrifugationHeatingThesimplestandtheleastexpensivemethodimprovethefeed’shandlingcharacteristicsmaypasteurizethefeedchiefconstraint

：thethermal

stabilityoftheproduct亮氨酸脫氫酶AdjustingpHReducethesolubilityoftheamphotericmolecules,suchasproteinandaminoacid.Controlmembranefoulingbychangingtheelectrostaticchargeofchargedcomponents.Cells,celldebrisandsomecolloidalparticlesareeasytoflocculateaslargeaggregates.例如，鏈霉素發(fā)酵液，酸化至pH3.0后，直接蒸汽加熱至70－75℃，維持30min的方法去除蛋白質(zhì)，能使過濾速度增大10～100倍，濾液粘度可降低1/6。CoagulationandflocculationElectricaldoublelayer雙電層

Coagulationandflocculationoccurinsuccessivestepsintendedtoovercometheforcesstabilizingthesuspendedparticles,allowingparticlecollisionandgrowthoffloc.agents：simpleelectrolytes

，polyelectrolytesElectricaldoublelayerElectricaldoublelayer

Innerlayer：sternlayer（吸附層或stern層）Outerlayer：diffuselayer（擴(kuò)散層）Why？Electrostaticinteraction靜電作用Thermalmotion熱運(yùn)動(dòng)Howcancolloidalstabilitybeachieved?electricaldoublelayerelectrostaticrepulsion靜電排斥hydrationshell(hydrationlayer)水化層Sternlayer：ionsofoppositechargearestronglybound

Diffuselayer：looselyassociatedwiththeobjectmadeoffreeionswhichmoveinthefluidSchematicofelectricdoublelayerSimpleelectrolytes（簡單電解質(zhì)）reducingtheelectrostaticrepulsion（靜電排斥）betweencolloidalparticles（膠體粒子）.

attractiveLondon-vanderwaalsforces（范德華引力）predominate.Thecolloidscanthencoagulateaslarger,denseparticleswhicharemoreeasilyfiltered.Al3+>Fe3+>H+>Ca2+>Mg2+>K+>Na+>Li+Al2(SO4)3·18H2O、AlCl3·6H2O、FeCl3等豆腐的制作石膏（CaSO4·2H2O）鹵水（MgCl2，CaSO4，CaCl2）過濾定型？？？Polyelectrolytesflocculantschemicalstructureofflocculantsmanyactivefunctionalgroupslongchainhighmolecularweightmechanism

adsorptionprovidebridgesclassification(accordingtofunctionalgroups)offlocculantsinfluencefactorsclassificationofPolyelectrolyteflocculantscationic（陽離子型）

maybeusedaloneneutralizethesurfacecharge

ofthecolloidalparticlesandreduceelectrostaticrepulsionorincombinationwiththealuminumandirontypecoagulants

anionic（陰離子型）oftenusedwithmetalcoagulantsnonionic（非離子型）oftenusedwithmetalcoagulantsinfluencefactorsmolecularweight,typeandamountofflocculantspHofthesolutionstirvelocityandstirtime右圖是α淀粉酶發(fā)酵液的絮凝試驗(yàn)中，絮凝劑加量對(duì)絮凝效果（濾速）的影響結(jié)果。filterAids助濾劑Adsorbcolloidsandreducecakecompressibility

吸附膠體，降低濾餅的可壓縮性Diatomaceousearth（硅藻土）orperlite（珍珠巖）

commonlyusedmethodsusedasaprecoataddedtothebrothbeforefiltrationselection選擇和使用助濾劑的要點(diǎn)（1）根據(jù)過濾介質(zhì)和過濾情況選擇助濾劑品種粗目濾網(wǎng)宜使用石棉粉、纖維素、淀粉；細(xì)目濾布宜使用細(xì)硅藻土；燒結(jié)或粘結(jié)材料宜使用纖維素。（2）粒度選擇當(dāng)粒度一定時(shí)，過濾速率與澄清度成反比。助濾劑的粒度必須與懸浮液中固體顆粒的尺寸相適應(yīng)。（3）使用量的選擇當(dāng)預(yù)涂時(shí)，間歇操作助濾劑的最小厚度為2mm；連續(xù)操作則要根據(jù)所需過濾率來確定。當(dāng)直接加入時(shí)，一般情況下，若用量與懸浮液中固形物含量相等，過濾速度最快。2.2Filtration過濾definitionFiltrationseparatessolidfromaliquidbyforcingtheliquidthroughasolidsupportorfiltermedium.classificationoperationbatchoperationcontinuousoperationclassificationmechanismclarificationfiltrationcakefiltrationMotivepowerforfluid

gravityfiltrationpressurefiltrationvacuumfiltrationcentrifugefiltrationBatchoperation（分批操作）UsefulforsmallfermentationbatchesFiltrationequipment

plateandframefilter（板框過濾機(jī)）Graduallyaccumulatesbiomass（生物量）,thenisopenedandclearedofthefiltercake廣泛應(yīng)用于培養(yǎng)基制備的過濾及霉菌、放線菌、酵母菌和細(xì)菌等多種發(fā)酵液的固液分離。推動(dòng)力來自泵產(chǎn)生的液壓或進(jìn)料貯槽中的氣壓比較適合于固體含量1％一10％的懸浮液的分離。板框過濾機(jī)優(yōu)點(diǎn)結(jié)構(gòu)簡單、裝配緊湊、過濾面積大；推動(dòng)力能大幅度調(diào)整，能耐受較高的壓力差（可超過0.1MPa,這是真空過濾器無法達(dá)到的）；固相含水分低，并能適應(yīng)不同過濾性質(zhì)的發(fā)酵液的過濾；輔助設(shè)備少、維修方便、價(jià)格低和動(dòng)力消耗少等。缺點(diǎn)設(shè)備笨重、間歇操作、勞動(dòng)強(qiáng)度大、衛(wèi)生條件差、輔助時(shí)間多和生產(chǎn)效率低。Continuousoperation（連續(xù)操作）Usedinlargerprocessestoreducedowntime（停工時(shí)間）filtersrotarydrumvacuumfilters真空轉(zhuǎn)鼓過濾器OperatingprincipleCharacteristicsapplicationcrossflowfiltration錯(cuò)流過濾diatomitefilter硅藻土過濾機(jī)OperatingprincipleDirectionofRotationKnifeDischargecakeFeedWashersWashingDryingLoadingRotaryDrumFilterCharacteristics自動(dòng)化程度高操作連續(xù)和處理量大特別適合于固體含量較大(＞10％)的懸浮液的分離受推動(dòng)力(真空度)的制約，一般不適合于菌體較小和粘度較大的細(xì)菌發(fā)酵液的過濾。過濾所得固相的干度不如加壓過濾。Crossflowfiltrationdead-endfiltrationthefeed(slurry)flowinthesamedirectionasthepermeateflow.Crossflowfiltration(tangentialflowfiltration)Fluidflowsparallel,orcrossflowtothefiltermediasurface.

Mainadvantage:theshearingactionoftheflowreducesthethicknessofthefiltercake.Membraneseparation:microfiltration,ultrafiltrationCharacteristics

CrossflowfiltrationCharacteristics不能得到干濾餅?zāi)芎谋纫话氵^濾高需要大的膜面積收率高（97-98%）濾液質(zhì)量好減少處理步驟對(duì)染菌罐批易于處理diatomitefilter右圖為垂直葉片式硅藻土過濾機(jī)適合過濾固形物含量<0.1%的懸浮液。2.3centrifugationdefinitionF,RCF,rpmTypesofcentrifugesLaboratorycentrifuges實(shí)驗(yàn)室離心機(jī)Preparativecentrifuges制備型離心機(jī)tubularbowlcentrifuge（管式離心機(jī)）disk/disccentrifuge（碟片式離心機(jī)）scroll/decantercentrifuge（螺旋式離心機(jī)）UltracentrifugationDifferentialcentrifugation差速離心Densitygradientcentrifugation密度梯度離心Ratezonalcentrifugation速率區(qū)帶離心Isopycniccentrifugation等密度離心Whenasuspensionisallowedtostand,thedensersolidsslowlysettletothebottomofthecontainerundertheinfluenceofgravity,aprocesscalledsedimentation（沉降）.Whenthissettlingisacceleratedwithacentrifugalfield（離心場）,theprocessiscalledcentrifugation.Centrifugationutilizesthedensitydifferencebetweenthesolidsandthesurroundingfluid.

Thesolidconcentrateproducedbycentrifugationdiffersfromthatproducedbyfiltration。Abestcentrifugationproducesapaste,oftenityieldsonlyamoreconcentratedsuspension.Filtrationproducesrelativelydrycake,whichisamajoradvantage.However,manybiologicalfeeds,whichcanbecentrifuged，cannotbeeffectivelyfiltered,sothatcentrifugationisoftenanattractivealternative.F,RCF,rpmCentrifugalforce

離心力Relativecentrifugalforce(RCF)

相對(duì)離心力Maximumrelativecentrifugalforce最大相對(duì)離心力CentrifugalforceF=mω2rF：thecentrifugalforceactingontheparticletomaintainitinthecircularpathm：themassoftheparticleω:theangularvelocityoftheparticler:theradiusofthepathRelativecentrifugalforce(RCF)RCF=G=F/W=mω2r/mg=(2πn)2r/gn：therotationalspeed,measuredinrevolutionsperminute（rpm）r：theradiusofrotationg：earth'sgravitationalaccelerationRCF=1.119×10-5×r×RPM2RPM,RCF,rMaximumrelativecentrifugalforce最大相對(duì)離心力實(shí)際工作中知道離心轉(zhuǎn)頭達(dá)到的最高速度以及該轉(zhuǎn)頭的最大直徑即可計(jì)算該轉(zhuǎn)頭工作產(chǎn)生的最大相對(duì)離心力。最大分離因素工業(yè)又把離心機(jī)能夠產(chǎn)生的額定最高RCF稱為最大分離因素。離心機(jī)能夠承受的RCF是離心機(jī)性能的重要參數(shù)。LaboratorycentrifugesRotorsFixedangledrotorSwing-outrotor（平拋式）Rmax-RminRmax-RminCentrifugetubePushbuttonDisplayrotorFrequentlyUsedLaboratoryCentrifugesBeckman-Coulter

FrequentlyUsedLaboratoryCentrifugesTubularbowlcentrifugesSuspensionisusuallyfedthroughbottom,andclarifiedliquidisremovedfromthetop.Solidsdepositonthebowl’swallasathickpaste.Thesuspensioncanbefeduntilsolidlossintheeffluent.Mostefficienthighcentrifugalforce（15000g－65000g）；thinsettlingzoneCanbecool,arealadvantageinproteinwork.

TubularbowlcentrifugesTubularbowlcentrifugesDisadvantageSmallsludgeholdingspace-onlyfordilutesuspensions(concentrationlowerthan1%byvolume)Solids

mustbemanuallyremoved.intermittentoperation（間歇操作）Thebowlmustbedismantled（拆卸）andcleaned.Applicationsseparationsofmicrobialcells，celldebris，cellorganelles，viruses，proteinsandnucleicacidsdiscDisktypecentrifugesDisktypecentrifugesFeedusuallyentersatthetop，

clarifiedliquidflowsoutanannularslitnearthefeed.types(accordingtothetypeofdischarge卸渣)Solidsejectingdisccentrifuge:intermittentoperationNozzledisccentrifuge:continuousoperationBasicdisccentrifuge:batchoperationMaximumRCF：1000g－20000gApplicationsseparationsofcelldebrisandmicrobialcells，suchasbacteria

andyeastscrolldischargecentrifugesThebowlandthescrewconveyorrotateinthesamedirection,butthelatterrotateswithavelocityslightlylowerorhigherthantheformer.FeedinClarifiedliquidSoliddischargescrolldischargecentrifugesthecontinuousfeeding,separatinganddischargingcanberealizedasitrotatesatitsfullspeed.Operationtemperature:upto300℃Canhandlesuspensionswithconcentrationsinthe1–50%range.applicationdewateringofbiologicalsludgesyeastandbacteriarecoverySummaryParametersTubularBowlDiskTypeScrollDischargeParticlesize0.01-100mm0.5-500mm0.5～2000mmParticleConcentration<1%<1-2%①<10%②<25%③1-50%RCF15000-650001000-200006000Processingcapacity0.1-0.4m3/h300m3/h0.4-60m3/hApplicationBacteria;celldebris,organelles;virus;proteinBacteria;yeast;Actinomycetes;CelldebrisInsulin;cytochrome;mycelia①人工卸料，②自動(dòng)間歇排料，③噴嘴連續(xù)排料DifferentialcentrifugationDifferentialcentrifugationDifferentialcentrifugation(differentialsedimentation)isaprocessofsuccessivecentrifugation(singleorrepeatedsteps)withincreasingcentrifugeforce.Separationisdependentonparticlemassandsize,whereheavierparticlesorcellssettlefirstatlowergvalues.Application:subcellularfractionation.Protein（蛋白質(zhì)）microsome（微粒體）Golgi（高爾基體）endoplasmicreticulumER（內(nèi)質(zhì)網(wǎng)）roughER,smoothERlysosome（溶酶體）peroxisome（過氧化物體）mitochondria（線粒體）nuclei，nucleus（細(xì)胞核）DifferentialcentrifugationAdvantagesEasytohandleSupernatant/Pelletiseasytoseparate.BigVolumefixedanglerotorDisadvantagesPoorPerformanceWalleffectDestroycellcomponentspellet（顆粒）nuclei，nucleus（細(xì)胞核）mitochondria（線粒體）lysosome（溶酶體）peroxisome（過氧化物體）microsome（微粒體）Golgi（高爾基體）endoplasmicreticulum，ER（內(nèi)質(zhì)網(wǎng)）roughER,smoothERDensitygradientcentrifugationDensitygradientcentrifugationutilizesaspecificmediumthatgraduallyincreasesindensityfromtoptobottomofacentrifugetube。Thismeansthatundercentrifugalforce，particleswillmovethroughthemediumanddensitygradientandstopatapointinwhichthedensityoftheparticleequalsthedensityofthesurroundingmedium.DensitygradientcentrifugationAdvantagesHighPerformanceWide

UsedKeepthePropertiesDisadvantagesTimeGradientBufferOperationExperienceRequiredDensitygradientcentrifugationCommonprocedures(1)preparegradient制備梯度(2)applysample加樣(3)Centrifuge離心(4)collectandanalyzefractions收集和分析組分DensitygradientcentrifugationIsopycniccentrifugationRatezonalcentrifugationRatezonalcentrifugation差速區(qū)帶離心法：當(dāng)不同的顆粒間存在沉降速度差時(shí)，在一定離心力作用下，顆粒各自以一定速度沉降，在密度梯度的不同區(qū)域上形成區(qū)帶的分離方法。Ratezonalseparationtakesadvantageofparticlesizeandmassinsteadofparticledensityforsedimentation.Gradientmaterial:sucrose，F(xiàn)icoll，PercollRatezonalcentrifugationCriteriaforsuccessfulratezonalcentrifugationDensityofthesamplemustbelessthanthatofthelowestdensityportionofthegradient.Densityofthesampleparticlemustbegreaterthanthatofthehighestdensityportionofthegradient.Thepathlengthofthegradientmustbesufficientfortheseparationtooccur.Time

isimportant.Ifyouperformtoolongruns,particlesmayallpelletatthebottomofthetube.Ratezonalcentrifugation100%separationispossiblewithasmallsample.Usedtopurifyenvelopedvirusesandtoseparatevarioustypesofmacromolecules

mixtureofproteins;differenttypesofRNA;separateDNAfromRNA,orproteins;ribosomalsubunits;polyribosomes;cellorganellesfromcrudecellularextracts.BioreactorfluidwithcellsmembraneVirusinactivationZonalcentrifugationConcentratedvirusUFmembrane

hydrolysisβ-propiolactoneβ-丙內(nèi)酯cellsproduct4℃37℃permeateNonantigenicmaterialFigure1.3downstreamprocessesforproductionofrabiesvaccine(狂犬疫苗)0.3μmTakes24hoursSucrosegradient10%-60%,50000g,15hIsopycniccentrifugationDuringthecentrifugation,theCsClgeneratesagradient(“self-generatinggradient”),andthemoleculesmovetothepositioninthegradientwheretheirdensityisthesameasthegradientmaterial.Separatesmoleculesbasedontheirdensity–mayhavethesamesize.因此用這種方法分離顆粒，主要是根據(jù)被分離顆粒的密度差異。只要被分離顆粒間的密度差異大于1%就可用此法分離。IsopycnicpositionIsopycniccentrifugationCriteriaforsuccessfulisopycnicseparation:Densityofthesampleparticlemustfallwithinthelimitsofthegradientdensities.Anygradientlengthisacceptable.Theruntimemustbesufficientfortheparticlestobandattheirisopycnicpoint(36-48hrs).Excessiveruntimeshavenoadverseeffect.IsopycniccentrifugationOftenusedtoseparatevarioustypesofDNA

circularvslineardoublestrandedvssinglestrandedDNAfromRNAhighlyrepetitiveDNA(satelliteDNA)fromotherDNAinthecellAlsocanbeusedforseparationoflipoproteinsandcellorganelles.MediumforIsopycniccentrifugationDensitiesofbiologicalmaterialMaterialDensity(g/cm3)Microbialcells1.05-1.15Mammaliancells1.04-1.10Organelles1.10-1.60Proteins1.30-1.40DNA1.70RNA2.00g/cm3蔗糖或者甘油(它們的最大密度是1.3g/cm3)通常可用于分離膜結(jié)合的細(xì)胞器，如高爾基體、內(nèi)質(zhì)網(wǎng)、溶酶體和線粒體。離心分離密度大于1.3g/cm3的樣品，如DNA、RNA，需要使用密度比蔗糖和甘油大的介質(zhì)。重金屬鹽氯化銫(CsCl)是目前使用的最好的離心介質(zhì)，它在離心場中可自行調(diào)節(jié)形成濃度梯度，并能保持穩(wěn)定。在氯化銫形成的密度梯度中，離心管頂部的密度為:1.65g/cm3，底部為:1.75g/cm3。因?yàn)镈NA的密度是1.70g/cm3，會(huì)停留在離心管的中部。QuestionsWhydoesfermentationbrothneedpretreatment?Whatarethesolutions?Howtouseafilteraid?Whatisthedifferencebetweencoagulationandflocculation?Howtousethemtogether?Howmanykindsoffiltrationmethodsarecommonlyusedinthebiologicalindustry?Describeitsprincipleandcharacteristicsbriefly.Whatarethebasicprinciplesandcharacteristicsofdifferentialcentrifugationanddensitygradientcentrifugation?Comparethedifferenceandrelationbetweenvelocityzonecentrifugationandiso-densitycentrifugation.Howmanykindsofcentrifugalmethodiscommonlyusedinbiologicalindustry?Describeitsprincipleandcharacteristicsbriefly.生物反應(yīng)器細(xì)胞收集細(xì)胞破碎提取純化產(chǎn)品制劑BioreactorExtractionRecoveryCelldisruptionCellharvestingPurificationProductformulationPrecipitationandcrystallizationSupernatantChapter3celldisruptionCellDisruptorsCellLysisCellHomognizingChapter3celldisruption3.1Cellsandcellwall/membrane3.2Celllysisbuffers3.3Celldisruptiontechniques3.3.1Mechanicalmethods3.3.2Non-mechanicalmethods3.4Efficiencyofcelldisruption3.1cellsandcellwall/membraneBacteriaGram-positivebacteriacell革蘭氏陽性菌細(xì)胞Gram-negativebacteriacell革蘭氏陰性菌細(xì)胞Fungalcell真菌細(xì)胞Yeastcell酵母細(xì)胞mouldcell霉菌細(xì)胞plantcellanimalcellCellwallsofbacteriaMaincomponentPeptidoglycan:apolymerconsistingofsugarsandaminoacidsthatformsamesh-likelayer.Cellwallstrengthdependontheextentofcross-linkingandthethicknessofthepeptidoglycanlayer.differatdifferentphasesandratesofgrowthasthedegreeofcross-linkageandthethicknessofthepeptidoglycanlayerincreasesascellsmovefromtheexponentialgrowthphase(對(duì)數(shù)生長期)tothestationaryphase（穩(wěn)定期）.溶葡球菌酶變?nèi)芫厝芫窷-乙酰－β－葡萄糖胺酶N-乙酰胞壁酸與N-乙酰氨基葡萄糖之間的β-1，4鍵FungalcellwallsThickness100to250nmStructure(frominsideout)achitinlayerChitinisthoughttomostlyaddstructuralstrengthtothecellwall.

alayerofβ-1,3-glucanalayerofmannoproteins葡聚糖合成酶幾丁質(zhì)葡聚糖甘露糖蛋白麥角固醇FungalcellwallsYeastcellwall

polysaccharides(80-90%),mainlymannans（甘露聚糖）

andglucans（葡聚糖）,withaminorpercentageofchitin.Glucansprovidestrengthtothecellwall,formingamicrofibrillarnetwork.甘露糖蛋白葡聚糖幾丁質(zhì)PlantcellwallsMainchemicalcomponentsCellulose（纖維素）Hemicellulose（半纖維素）Pecticsubstances:pecticacid（果膠酸）,pectin（果膠）,protopectin(原果膠)Lignin（木質(zhì)素）StructureIntercellularlayerPrimarywallSecondarywallcelluloseGlucoseCelluloseMicellae（微團(tuán)）Microfibril（微纖絲）Macrofibril（大纖絲）SummaryBacteriaFungalPlantG+G-YeastFungalCellWallThickness20-80nm10-30nm70-300nm100-250nm500-700nmMaincomponentPeptidoglycanPeptidoglycanlipidpolysaccharides(MannansGlucans)chitinlayerβ-1,3-glucanmannoproteinsCelluloseHemicellulosePecticsubstancesCellwallstrength

++++++++++++3.2celllysisbuffersionicstrength0.1~0.2mol/L；pH7.0~8.0Tris-HClbuffer

Phosphatebuffer（磷酸緩沖液）Antioxidants

DTT（Dithiothreitol

，二硫蘇糖醇）β-mercaptoethanol（β－巰基乙醇）Cysteine（半胱氨酸）reducedglutathione(GSH，還原型谷胱甘肽)3.2celllysisbuffersProteaseinhibitorsSerineproteaseinhibitors:DFP,PMSF,TPCKThiolproteaseinhibitors:Iodoaceticacid(碘乙酸)Metalprotease/metalloproteaseinhibitor:EDTASubstratesorcofactorsofenzymesPVP(polyvinylpyrrolidone，聚乙烯吡咯烷酮

)

3.3.1MechanicalmethodsMechanicalgrinding機(jī)械研磨

BeadMill珠磨機(jī)Homogenization勻漿

highpressurehomogenization高壓勻漿Ultrasound（sonication）超聲波X-pressFrenchpressDisadvantagesofMechanicalDisruptionBeadmillMechanismsofcelldisruptionProcessvariablesofabeadmillListsomeoftheparametersthataffectthedisruptionefficiencyofabeadmillCharacteristicsExperimentationMechanis