生物工程下游技术-第五章

Separationandpurificationofproducts,Chapter5Celldisruption,proteinrecoveryandseparation,第五章细胞破碎、蛋白质复性和固液分离,Inthischapter,Weconsiderthemethodsforcellrupturemostlikelytobeusefulatlargerscales.Thesemethodsareconvenientlysplitintotwogroups,describedas“chemical”and“mechanical”.Inthechemicalmethods,thecellmembraneisrupturedbyosmoticpressure,dissolvedbydetergents,orenfeebledbyorganicsolvents.Thesechemicalcasesareoftengentlesothattheproductsarenotirreversiblydenaturedevenwhilethecellsareruptured.Scale-upiseasy:Ifwewanttotreat10timesthebiomass,weneedtouse10timesasmuchchemical.,Themechanicalmethodsaredominatedbyhomogenizationorballmillgrinding.Inbothcases,cellsarebrokenbyhighmechanicalshear.Heatgenerationisaproblem.,TABLE1.ChemicalCellDisintegrationTechniques,表1.细胞化学破碎法,TABLE2.MechanicalCellDisintegrationTechniques,表2.细胞物理破碎法,CHEMICALMETHODS,Thechemicalmethodsofcellrupturearedominatedbyosmoticshock,detergentsolubilization,andlipiddissolution.Thesethreemethodsaredescribedinthefollowingparagraphs.Beforethisdescription,wewanttogivesomebriefgeneralizationsabouttheothertwomethods:enzymedigestionandalkalitreatment.,Bothenzymedigestionandalkalitreatmentareeffectivemethodsofcellrupture,buteachhasamajordisadvantage.,ThedisadvantageofenzymedigestionisthecostoftheenzymeswhichFrequentlymakesthismethodprohibitiveatalargescale.Thisisunfortunate,becausethemethodisbothgentleandselective.Itisnothingmorethanaddingtoacellsuspensionenzymeswhichreactquicklywiththecellwallsanddestroythem.Becausetheenzymesselectivelycatalyzethesecellwallreactions,theyreactonlysparinglywithothersoluteswithinthecell.,Alkalitreatmentistheantithesisofenzymedigestion,foritisharshratherthangentle,nonselectiveratherthanspecific,andcheapratherthanexpensive.Alkaliaddedtoacellsuspensionreactswiththecellwallsinanumberofways,includingthesaponificationoflipidsinthecellwalls.,Inasense,thetreatmentisanextremeexampleofsolubilization,foritconvertscellwallcomponentsintodetergents.Includingproteindenaturations.Asaresult,themethodtendsbothtorupturecellwallsandtodestroyproducts.Eventhoughitischeap,alkalitreatmentislessusefulthanthethreemethodswhicharedetailednext.,A、OsmoticShock,Thesimplestofthethreemajorchemicalmethodsisosmoticshock.Thisisnothingmorethandumpingagivenvolumeofcellsintopurewater-oftenabouttwicethevolumeofcells.Thecellsswellbecausetheycontainsoluteswhichcauseanosmoticflowofwaterintothecells.Insomecases,theyswellsomuchthattheyburst.Theircontents,releasedintothesurroundingsolution.,Thesusceptibilityofcellstolysisdependsstronglyontheirtype.Redbloodcellsareeasilylysed.Animalcellsoftencanbelysed,butonlyafteranimaltissuehasbeenmechanicallymincedorhomogenized.Plantcellsaremuchmoredifficulttolyse,fortheircellwallsoftencontainstrongwoodymaterialwhichisrelativelyimpermeabletoosmoticflow.,B、Solubilization,Thesecondmajormethodofchemicallyrupturingcellsissolubilizationbydetergents.Typically,aconcentrateddetergentsolutionisaddedtoabouthalfthesolutionsvolumeofcells.Thedetergentdisruptsthecellmembrane.Theresultingsuspensioncanbecentrifugedtoremovecellfragments,andthenrunthroughanadsorptioncolumnoranextractortoisolatetheproduct.,5.Large-ScaleRecoveryofProteinInclusionBodiesbyContinuousCentrifugation,Inclusionbodies(IBs)aremicron-sizedsolidproteinparticlesthatformwithinthecytoplasmofcertainhostcellssuchasEscherichiacolifollowingoverexpressionofaprotein.IBsarecomprisedprimarilyoftherecombinantproteinofinterest.Somecontaminantsincludingnonproductprotein,nucleicacids,andcell-envelopecontaminantscanalsobeincorporatedintothegranules.,However,itisbelievedthatthemajorityofcontaminantsactuallyadheretotheIBsurfacefollowingreleasefromthecytoplasmduringprocessing(1).ThisindicatesthatIBformationinvivoisaratherspecificprocessthatofferscertainadvantagesfordownstreamprocessing.Specifically,theproteinofinterestalreadyexistsinarelativelypurestateasasmallgranulethatcanberecoveredbyphysicalseparationfromnonassociatedcontaminants.Ofcourse,ifasuitablyefficientproteinrefoldingstrategyisnotavailable(2),thenanygainsachievedthroughinclusionbodyformationmaybeeasilylost.,Strategiesforthelarge-scaleprocessingofproteinsformedasIBswithinE.coliarehighlyconservative,andbasedlargelyonlaboratoryprocedures.IBsareinitiallyreleasedfromthecellbymechanicaldisruption,providingamixtureofsolidcellulardebris(cell-wallparticles,andsoon),solublehostcontaminants,andtheIBs.,ContinuouscentrifugationisthenemployedtoseparatethedenserIBsfromcontaminants.Continuouscentrifugationdiffersfromlaboratorycentrifugation,asthemachinescanbeoperatedtoprovideadegreeofseparationofthesolidIBsandtheparticulatecelldebris.Followingcollection,andusuallywashing,theIBsaredissolvedinstrongdenaturantpriortoproteinrefoldingandrecoverybyhigh-resolutionmethodssuchaschromatography.Furtherdescriptionoftypicalprocessesisprovidedelsewhere(3,4).,IBrecoverydependsstronglyonthecentrifugedesignandoperationalparameters.Themostcommonlyemployedcentrifugeforlarge-scaleIBcollectionisthedisk-stackdesign(Fig.1).Materialisfedthroughthecenterofthediskstacktotheouterperiphery,whereitisthenforcedbetweenthediskswhicharerotatingatahighspeed.,Solidsarecollectedonthedisksandflungtotheouterperipherybycentrifugalforce.Asludgeofparticles(IBsandsomedebris)iscollectedinthebowlperiphery,whereitcanbedischarged(seeNote1).Clarifiedsupernatantflowsbetweenthedisksandexitsatthemachinecentre,carryinganyuncollectedparticles.,Essentiallycontinuousseparationofsludgeandsupernatantisthereforeachieved,withouttheneedtodisassemblethecentrifugeforsolidsremoval.Machinesrangingfromlaboratorysize(e.g.,200mL/minoffeedsuspension)throughtofull-scaleproductionareavailablefromthetwomainmanufacturers,namelyWestfaliaSeparatorAG(Oelde,Germany)andAlfaLavalSeparationAB(Tumba,Sweden).,Fig.1.Crosssectionofatypicaldisk-stackcentrifugeforinclusion-bodycollection.ReproducedwiththekindpermissionofWestfaliaSeparatorAG,Oelde,Germany,fromthebooklet“Centrifugalclarifiersanddecantersforbiotechnology.”,workingprinciples,howadiskstackcentrifugeworks,1、目标产品分离与纯化的意义目标产品的分离纯化在现代生物技术工业中占十分重要的位置，它决定着产品的纯度和安全性，也决定着产品的收率和成本。2、分离纯化的两个基本阶段1）产物的初级分离阶段2）产物的纯化精制阶段,第一节概述,初级分离阶段一般位于生物反应之后，其任务是分离细胞和培养液，破碎细胞释放产物（如果产物在胞内），溶解包涵体，复原蛋白质，浓缩产物和去除大部分杂质等。纯化精制阶段是在初级分离的基础上，用各种高选择性手段（其中主要是各种色谱层析）将目标产物和干扰杂质尽可能地分开，使产物的纯度达到有关的要求，最后制成可以储藏、运输和使用的产品。,悬浮液的共同性质,1、目标产物浓度普遍比较低，悬浮液中大部分是水2、组分非常复杂，是含有细胞、细胞碎片、蛋白质、核酸、脂类、糖类、无机盐类等多种物质的混合物。3、分离过程很容易发生失活现象，PH、离子强度、温度等变化常常造成产物的失活。4、性质不稳定，易随时间变化，如受空气氧化，微生物污染，蛋白质水解作用等。,分离要做到什么？,1、迅速加工，缩短停留时间2、控制好操作温度和PH值3、减少或避免与空气接触受污染的机会4、设计好各组分的分离顺序,第二节细胞破碎,细胞破碎技术是指利用外力破坏细胞膜和细胞壁，使细胞内容物包括目的产物成分释放出来的技术，是分离纯化细胞内合成的非分泌型生化物质（产品）的基础。结合重组DNA技术和组织培养技术上的重大进展，以前认为很难获得的蛋白质现在可以大规模生产。,细胞破碎机理图,1、自20世纪80年代初重组DNA技术得到广泛的应用以来，生物技术发生了质的飞跃，产品的数量越来越多，许多具有重要应用价值的产品应用而生（胰岛素、干扰素、生长激素、白细胞介素等）。2、细胞破碎是提取胞内产物的关键步骤，它影响产物的活性、收率和成本，因而引起基因工程和生化工程学者的关注。,细胞破碎技术的发展背景,细胞破碎技术很多，可以用渗透原理，让细胞吸水涨大而达到细胞膜破碎的目的。例：用纯化水稀释血液也可以用离心的原理让细胞破碎，如将血液放入离心机中在4000转/分的转速下离心3分钟。需注意的是细胞破碎用的是细胞而不是组织或其他。,细胞破碎阻力,细菌几乎所有细菌的细胞壁都是由肽聚糖（peptidoglycan）组成，它是难溶性的聚糖链（glycanchain），借助短肽交联而成的网状结构，包围在细胞周围，使细胞具有一定的形状和强度。短肽一般由四或五个氨基酸组成，如L-丙氨酰-D-谷氨酰-L-赖氨酰-D-丙氨酸。而且短肽中常有D-氨基酸与二氨基庚二酸存在。破碎细菌的主要阻力是来自于肽聚糖的网状结构，其网结构的致密程度和强度取决于聚糖链上所存在的肽键的数量和其交联的程度，如果交联程度大，则网结构就致密。,酵母菌,酵母细胞壁的最里层是由葡聚糖的细纤维组成，它构成了细胞壁的刚性骨架，使细胞具有一定的形状，覆盖在细纤维上面的是一层糖蛋白，最外层是甘露聚糖，由1,6一磷酸二酯键共价连接，形成网状结构。在该层的内部，有甘露聚糖-酶的复合物，它可以共价连接到网状结构上，也可以不连接。与细菌细胞壁一样，破碎酵母细胞壁的阻力主要决定于壁结构交联的紧密程度和它的厚度。,真菌,霉菌的细胞壁主要存在三种聚合物，葡聚糖（主要以-1,3糖苷键连接，某些以-1,6糖苷键连接），几丁质（以微纤维状态存在）以及糖蛋白。最外层是-和-葡聚糖的混合物，第2层是糖蛋白的网状结构，葡聚糖与糖蛋白结合起来，第3层主要是蛋白质，最内层主要是几丁质，几丁质的微纤维嵌入蛋白质结构中。与酵母和细菌的细胞壁一样，真菌细胞壁的强度和聚合物的网状结构有关，不仅如此，它还含有几丁质或纤维素的纤维状结构，所以强度有所提高。,植物细胞,植物细植物细胞可以分为初生壁和次生壁。初生壁是细胞生长期形成的。次生壁是细胞停止生长后，在初生壁内部形成的结构。目前，较流行的是由Lampert等人提出来的经纬模型。依据这一模型，纤维素的微纤丝以平行于细胞壁平面的方向一层一层敷着在上面，同一层次上的微纤丝平行排列，而不同层次上则排列方向不同，互成一定角度，形成独立的网络，构成了细胞壁的“经”，模型中的“纬”是结构蛋白（富含羟脯氨酸的蛋白），它由细胞质分泌，垂直于细胞壁平面排列，并由异二酪氨酸交联成结构蛋白网，径向的微纤丝网和纬向的结构蛋白网之间又相互交联，构成更复杂的网络系统。,一、细胞破碎技术的分类,目前已发展了多种细胞破碎方法，以便适应不同用途和不同类型的细胞壁破碎。破碎方法可规纳为机械法和非机械法两大类。,常用破碎方法,二、几种常用的破碎方法,采用高压匀浆器（由高压泵和匀浆阀组成，英国APV公司和美国Micr