生物分离工程-双语版-2讲课稿.ppt

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1、生物分离工程-双语版-21 IntroductionvProtein folding v(1)short ranged interaction(secondary structure)v(2)gradual combination and reshuffling(重新组合）(subdomains,domains,and subunit assemblies)Second generation biotechnological productsv(1)larger moleculesv(2)more complexv(3)more glycosylatev(4)more sulphydryl b

2、onds1 IntroductionvInitial bioseparation importancev(1)filtrationv(2)centrifugationv(3)chromatographyv Classified heuristics（论据，规则）v(1)unit operation methodsv(2)design step sequence v(3)species(components properties)v(4)composition 1 IntroductionvThree factors of designing bioseparation processesv(1

3、)purityv(2)costv(3)marketvMaterial state and choice of separation methodsvMaterial secreted(ultrafiltration,centrifugation)vNot secreted material(cell disruption,solid-liquid separation)vMaterial in liquid(ultrafiltration,adsorption)vMaterial in solid(extraction into aqueous solution)New and words v

4、Protacte lawsuits 诉讼vDeleterious 有害的，有毒的vVexing 烦恼vHierarchic 分等级vSubdomain 亚域vDomains 蛋白质结构的域vSubunit 亚单位vEndoplasmic reticulum 内质网vDisulfide bond 二硫键vAcademia 学术界vDelicate 脆弱的vFlowchart 流程图vCytoplasmic 细胞质的vPeriplasmic 外周胞质vOsmotic 渗透v urea 脲vGuanidine 胍II Product excretion from the cell or cell d

5、isruption vExcreted protein advantagev(1)contamination level lowv(2)easy downstream processing stepsv(3)not require cell disruptionv(4)avoid handling viscous fluids and proteinase releasev(5)facility extraction of products v(6)signal peptide determines secretionCell disruption v(1)energy-intensive(强

6、烈）and violentv(2)without destroying interest proteinv(3)contain(兼容）process(bacteria,v yeast,mammalian,plant,and insect v sources)v(4)be controlledv(5)be validatedProducts requiring cell disruptionvVaccines(tetanus(破伤风）,meningitis（脑膜炎)vGlucokinase（葡萄糖激酶）vGlycerokinase（甘油激酶）vInvertase（转化酶）vToxin(enter

7、otoxin)vSubcellular components(mitochondrai,chloroplast)vIntracellular constituents(DNA,RNA preparation,and virus-like particles)vRecombinant insulinvRecombinant growth hormonevProtein A and G.Ideal large scale cell disrupterv(1)disrupt tough organismsv(2)mechanism of cell disruption should be well

8、understoodv(3)sterilizablev(4)containable（兼容）and validatablev(5)amenable to being made automaticv(6)continuous and compact（紧凑的）v(7)economicalCell disruption goalvObjective function(monetary（金钱）value of v product)vSmall scale cell disruptionv(1)ultrasonicv(2)freeze thawing(熔化）v(3)force cells through

9、very small orifices at high pressurev(4)glass or porcelain ball millsLarge scale cell disruptionvHomogenizer vStructure(positive displacement pump,pistons,nozzle)vMechanisms v(1)Shearv(2)Cavitation（气穴）v(3)impingement(冲击）vConditionv(1)concentration(4-175 g dry wt/liter)v(2)pressure(30-95 MPa)v(3)pass

10、es number(5time)v(4)modeled by equation v(5)wild-type E.coli cells more difficult to disrupt(heat induction at 42C)Ln(1/(1-R)=KNPDelineating mechanisms of cell disruptionv(1)facilitate controllingv(2)validatingv(3)reproduciblev(4)better design and optimize equipmentv(5)Turbulent eddies(湍流涡旋）Responsi

11、ble for cell disruption oscillate)v(6)shear stress,turbulence by v impingementEnzymatic lysis cell disruptionv(1)a lytic protease(dissolve the outer part of the cell wallv(2)endo-(l-3)glucanase（葡聚糖酶）to disintergrate glucan netv(3)source（酶的来源）and culture system kinetic analysisvA understanding the sy

12、stemvB designing the small and large scale systems vC cytophaga（吞噬细胞）system exhibits high protease activity,higher conversion and higher lysisrate at longer timevD oerskovia systems high glucanase low protease activity,contain some inhibitorsvE an initial lag in yeast lysis by both enzymevF simple t

13、wo step model(cell mass solubilized,protein hydrolyzed to peptides)vG Mishaelis-Menten kinetic equation predict cell wall lysisvH production of food grade single cell protein New words and vocabularyvAmylase 淀粉酶vModulate 调节vInterferons 干扰素vViscous fluids 粘性流体vSignal peptide 信号肽vCarboxypeptidase 羧肽酶v

14、Alkaline phosphatase 碱性磷酸酯酶vGlyceraldehyde-3-phosphate dehydrogenase 甘油醛-3-磷酸脱氢酶vReduced nicotinamide adenine 还原型尼克酰胺腺嘌呤vDinucleotide 二核苷vReductase 还原酶vHydrophobic 疏水的vHydrophilic 亲水的New words and vocabularies(2)vPathogens 病源体vBall mills 球磨机vResonance 共鸣，回声vVibration 振动，摆动vDissipate 分散vPinpointing 最

15、佳点,针尖vOscillate 振荡vCandida utilis 假丝酵母vExtrusion 喷出vSapphire 兰宝石vJewel 宝石vThermocouple 热电偶vSleeve 袖子，套vHydrodynamic 流体力学vHydroxylase enzyme 羟化酶vSynergistic 增效，协同作用vGlucan 葡聚糖vMorphology III initial fractionationvPrimary isolation stepv(1)adsorptionv(2)extractionv(3)precipitationv(4)distillationv(5)f

16、iltration v(6)solvent evaporationvRequirementvSignificant volume reductionInterest product in solutionv(1)gradually remove impuritiesv(2)increase product concentrationv(3)chose methods depends on scale and organismv(4)washing stages followed by dewatering stepvYeasts easy to deal withv(1)10 m in dia

17、meter v(2)easily flocculatedv(3)centrifugation v(4)filtrationv(5)cross-flow filtrationAnalysis disrupted bakers yeast suspensionsv(1)viscosityv(2)densityv(3)sedimentationvMeaning v(1)minimize degradation cell wall following rupturev(2)minimize cell fragment proportionv(3)restrict viscosity increasev

18、(4)correlation between lab and pilot scale centrifuges v or industryv(5)estimate separation costs at an early stageFirst crude separation or final product dewater steps v(1)adsorptionv(2)precipitationv(3)membrane separation v A cross flow filtrationv B microfiltrationv C ultrafiltrationv(4)liquid-li

19、quid extraction Aqueous two-phase partitionv(1)polymer+salt,polymer+polymerv(2)easy to scale upv(3)biocompatiblev(4)rapid(with centrifugation)v(5)purification and concentration of biological productv(6)cell particles and nucleic acids removedv(7)large volume reductionA adsorptionv(1)purify proteinv(

20、2)difficult to removing particulate using packed or fixed bedsv(3)particulate entrapped in voids of the bedsv(4)excessive pressure drops and deformation of adsorbentv(5)expanded bedv(6)fluidized bedExpanded bed adsorptionv(1)eliminate a step for removal of cell or debrisv(2)scale up simple(similar t

21、o packed bed)v(3)combine clarification,concentration and purificationv(4)could be used for viscose fluidv(5)high biomass content of feedstockv(6)expanded bed stabilized by wide distribution of particle sizeNew words and vocabularyvColloidal（胶状的）vGlycan（聚糖）vLysate（溶解物）vAerosol（悬浮微粒,气溶胶）vParticulate（微

22、粒）vFluidized bed（沸腾床）vStreptomycin（链霉素）vNovobiocin（新生霉素）vAnnexin V（膜联蛋白一类被钙离子活化后可与膜磷脂结合的蛋白）Scout（侦察）vWashing （洗涤）vElution（洗脱）New words and vocabularyvReverse phase high pressure liquid phase chromatography(反相HPLC）vCircumvented（包围）vHybridoma（杂合细胞，(淋巴细胞)杂交瘤）vPenicillin（青霉素）vTetracycline（四环素）vCephalosp

23、orin C（头孢菌素C）vDivinyl benzene（二乙烯苯）vStyrene（苯乙烯）B membrane separationvFunction:Clarification and sterilizing vProblems v(1)Solid-liquid interface adsorptionv(2)significant surface area(500cm2)vMembrane materials v(1)hydroxyl-modified hydrophilic polyamide v(2)hydrophilic polyvinylidene fluoride(PVDF

24、)聚偏二乙烯氟化物）聚偏二乙烯氟化物）v(3)nylon-66vInterest protein v(1)insulin(100%recovery)v(2)bovine serum albumin(BSA)98%recoveryv(3)immunoglobulinMinimizing protein lossv(1)low-binging protein membranev(2)dilute protein solutionv(3)expensive v(4)examine prefilters and final filtersRemove of virusv(1)thermal inact

25、ivationv(2)chemical treatmentv(3)denaturant(urea)v(4)-propiolactone（丙内酯）-UV light exposurev(5)membrane filtrationv A base on sizev B reproduciblev C predictable v D completev E validatable v F high protein recovery New words and vocabularyvInsulinvBovine serum albumin（牛血清蛋白）vImmunoglobulin（免疫球蛋白）vSc

26、ale-down（按比例缩减）vDiscernible（可辩别的）vSteady state（稳态）vAsymptotically（渐近的）vLow-binding protein membranev-propiolactone UV light（-丙内酯-紫外光）vMonotonically（无变化的，单调的）vPlatelet（血小板）vImmunodiseasevHydraulic permeability（水渗透性）vFiltration resistance（过滤阻力）vPure water permeability(PWP)纯水透过性Vocabularies Domain disu

27、lfide bonds,signal peptide,turbulent eddies Aqueous two-phase partitioning expanded bed adsorption fluidized bed packed bed reverse-phase high pressure liquid-phase chromatography floculate biocompatible scaled-down steady-state langmuir isothermQuestions v1 Which advantages secreted proteins?v2 Whi

28、ch differences between Mammalian cell proteins v and microorganism proteins?v3 Which methods disrupting cell in laboratoryv4 What is the most important in early part of v bioseparation?v5 What advantage have expanded bed adsorption by v comparing with packed bed?v6 How to remove virus or virus-like

29、in protein solution?Chapter 2 SecC UltrafiltrationvExample：antibodies productionv(1)antibodies applicationvA diagnosis vB affinity ligand(cytokines and blood-clotting factors)v(2)membrane material(cellulose triacetate)v(3)use v A 50-100-fold concentrationv B minimizing shear forcesv C tangential and

30、 frame ultrafiltration system v D gel polarization（浓差极化）reducedv F acceptable flux rateDenaturation during ultrafiltrationv(1)conformational changev(2)instabilityv(3)immunogenicity(免疫原性）vInfluencing ultrafiltration factorsvA electrostatic interaction(attraction,repulsion)vB Hydrophobicity New words

31、and vocabularyMandate（命令，要求）Diagnostics 诊断学Cytokines 细胞因子Blood clotting factors（凝血因子）Tangential plate 切向板，错流过滤板Undermine（破坏）Integrity（完整）Tryptic-casein peptides-酪蛋白肽Antihypertensive（抗高血压的）Immunomodulatory 免疫调节剂Plasmin 血纤维蛋白溶解酶Transmission 输送Detrimental 有害的Interfacial tension 表面张力Emulsion liquid memb

32、rane 乳状液膜Carrier 载体Diffusivity 扩散率D cross-flow filtrationvAffinity cross-flow filtrationv(1)combining bioadsorption and membrane in a single stepv(2)escort supportsv A chemically and mechanically stablev B possess a large specific areav C Negligible nonspecific adsorptionv D easily modifiable to inc

33、orporate ligandsv(3)high protein loss in washing stepNew words and vocabulariesvAffinity cross-flow filtrationvEscort 簇vSupernatant 上清液vMonomolecular layervInnermost 内心的，最里面的vLangmiur isothermvGlutaradehydevSchiff basesvSodium borohydridevMicroparticles E Emulsion liquid membrane vPenicillin G extra

34、ction by emulsion liquid membranevApplicationvConcentrate weak acids,bases,metal ion and biomaterial vPenicillin extraction v(1)Carrier:aminev(2)pH adjuster Na2CO3 at pH 5-8,penicillin G v stablev(3)9 times concentrationF ExtractionvInsulin purification processv(1)extraction by ethanolv(2)acidificat

35、ion at pH 2v(3)neutralization with calcium carbonatev(4)vacuum concentrationv(5)salt precipitationv(6)reprecipitation by isoelectric pointv(7)gel filtration chromatography or ion exchange chromatographyvDifficult information availablev(1)effect of different process variablesv(2)sequencing of differe

36、nt subprocessesv(3)scale level of operationNew words and vocabulariesvEmulsion liquid membrane 乳化液膜vAmine 胺vCarrier 载体vPancreas 胰腺vHyperglycemia 高血糖vDiabetes mellitus 糖尿病vPorcine origin 猪器官vIsoelectric point 等电点vReverse-phase chromatography 反相层析vScarce 缺乏的，不足的vProprietary 所有权G precipitation vApplyin

37、g high resolution technique at earlier stage v(1)reduce volumev(2)reduce chemical consumptionvAffinity precipitation using chitosan as a ligandvPrinciple vSoluble ligand binding to target protein lead to precipitatev(1)aggregatev(2)heterobifunctional ligandsvIsolating trypsin with STI chitosan Preci

38、pitation factorsv(1)salt and cosolvents(supersaturation,seed particles,crystallization,agglomerationv-lactalbumin separationvWhey components:lactose,proteins,salts and residual fatv(1)1:1 binding calcium ion(-1A)v(2)conformational change(unfolding transitions,surface hydrophobicity)v(3)selectively s

39、eparating lactalbumin(-1A)v and-lactoglubulin at pH isoelectric point and above 50 v(4)nonspecific precipitationAffinity precipitation using ligand modified phospholipid as a ligand carriervPrinciple vSoluble ligand binding target protein to form precipitatevHeterobifunctional ligand(binding target

40、molecule and facilitate precipitation of liand-target protein)vBiotin ligand binding avidinv(1)rapid bindingv(2)slow hydrophobic interaction between nonpolar alkyl side chains of phospholipidsv(3)high yield and purity of avidinvDrawbacksv(1)difficult to obtain stable and inexpensive bis-ligands,poly

41、ligands and ligand carriersv(2)adsorption capacity lowv(3)nonspecific adsorption degree high Affinity precipitation mechanismvTwo mechanism v(1)primary effect vProtein cross-linking and forming network with ligandv(2)second effect vSolubility decrease of ligand carrier due to pH,temperature,and salt

42、 concentrationSTI immobilized on PLS as an affinity agent for trypsin precipitationv(1)material v A artificial solution containing BSA and trypsinv B a crude pancreatic extractv(2)salt precipitated PLS due to osmotic shock v(3)PLS redispersed in solution after removing salt by centrifugev(4)PLS had

43、large specific surface area and ligand coupling capacityv(5)lack of nonspecific adsorptionv(6)91%trypsin coprecipitated in artificial solution and 87%trypsin recovered v in precipitate by 0.01 M NaOHv(7)STI-PLS repeated three timesv(8)Purification factor was about six v(9)loss of binding capacity(93

44、.3%,87.7%,83.3%)v(10)80%activity recovery and specific activity with 1X104 U/mgv(11)precipitate and redisperse 10 times by added saltNew words and vocabulariesvAffinity precipitation 亲和沉淀vHeterobifunctional ligand 异种双功能试剂vChitosan 壳聚糖vOligomeric 寡聚体的vSoybean trypsin inhibitor 大豆蛋白酶抑制剂vChymotrypsin 胰

45、凝乳蛋白酶，糜蛋白酶vSupersaturation 过饱和v-lactalbumin 乳白蛋白vWhey protein 乳清蛋白vMammary gland 乳腺vTrytophan 脯氨酸vTransitions 过渡vTurbidity 混浊vSurfactant 表面活性剂vPhospholipids 磷脂vAvidin 生物素结合蛋白vBiotin 生物素vDimyristoylphosphatidylethanolamine 二肉豆蔻酰磷酸乙醇胺vOctaethylene glycol mono-n-dodecyl-ether 八乙烯醇单十二烷基醚vHastened 促进，加速v

46、Polymerized liposome 聚合脂质体vRedispers 再分散vSteric hindrance 空间位阻H chromatographic proceduresvLarge scale purificationv(1)yieldv(2)purityv(3)operation scalev(4)chromatography(limited extent in initial step)vExample 2.11 vStaphylococcal enterotoxin B purification using chromatographyv(1)Two ion exchange

47、 steps v(2)One gel filtration stepv(3)high linear flow rates at low pressure 5 kPv(4)culture supernatant v A(centrifugation,sterile filtration,adsorption).v B content 0.4mg/Lv C protein content 11mg/mlv D purity 0.004%v E volume 400 LPharmacia column 370 KS purificationv(1)protein content from 11 to

48、1.5mg/mlv(2)SEB content from 0.00042 to 0.12 mg/mlv(3)purification factor 2000vBiological molecules product standardsv(1)DNA 10 pg/dosev(2)no virusesv(3)pyrogen 300 endotoxins IU/doseExample 2.12vDivinylbenzene-polystyrene resin separation v(1)analysis of thermodynamics using HPLCv(2)linear adsorpti

49、on isotherm above 293-313Kv(3)temperature-dependent adsorption isotherm constants S,Hv(4)pore size diameterv(5)polarity of resinv(6)chromatography is only a choice for industrial separationNew words and vocabulariesvStaphylococcal enterotoxin 葡萄球菌外毒素vMethicillin 甲氧苯青霉素vPharmacia columnvDose 剂量vPyrog

50、en 热原vMiniscule 小字的vDivinylbenzene polystyrene copolymer 苯乙烯-二乙烯苯共聚物vAspartame 门冬氨酰苯丙氨酸甲酯（比蔗糖甜200倍的甜味剂）vPhenylalanine 苯丙氨酸vAspartic acid 天门冬氨酸vAsparagine 天门冬素，天冬酰胺酸vAdsorption isotherm 吸附等温线vHomologues 类似物，同族体IV high-resolution fractionationvPrevious purification steps v(1)volume reductionv(2)clarif