生物分離工程雙語版2

1、1 IntroductionvConsidering protein properties for bioseparationv(1) sizev(2) chargev(3) biological affinityv(4) hydrophobicity-solubilityvProducts from mammalian cell techniquesv(1) enzymev(2) hormonesv(3) nonantibody immunoregulatorsv(4) monoclonal antibodiesv(5) tumor specific antigens1 Introducti

2、onvProtein folding v(1) short ranged interaction ( secondary structure )v(2) gradual combination and reshuffling (重新組合） (subdomains, domains, and subunit assemblies)Second generation biotechnological productsv(1) larger moleculesv(2) more complexv(3) more glycosylatev(4) more sulphydryl bonds1 Intro

3、ductionvInitial bioseparation importancev(1) filtrationv(2) centrifugationv(3) chromatographyv Classified heuristics（論據(jù)，規(guī)則）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) p

4、urityv(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 an

5、d words vProtacte lawsuits 訴訟vDeleterious 有害的，有毒的vVexing 煩惱vHierarchic 分等級vSubdomain 亞域vDomains 蛋白質結構的域vSubunit 亞單位vEndoplasmic reticulum 內質網(wǎng)vDisulfide bond 二硫鍵vAcademia 學術界vDelicate 脆弱的vFlowchart 流程圖vCytoplasmic 細胞質的vPeriplasmic 外周胞質vOsmotic 滲透v urea 脲vGuanidine 胍II Product excretion from the cell

6、or cell disruption 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) en

7、ergy-intensive(強烈） 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(破傷風）, meningitis（腦膜炎)vGlucokinase（葡萄糖激酶）vGlycerokinase （甘

8、油激酶）vInvertase （轉化酶）vToxin (enterotoxin)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) mech

9、anism of cell disruption should be well understoodv(3) sterilizablev(4) containable（兼容） and validatablev(5) amenable to being made automaticv(6) continuous and compact（緊湊的）v(7) economicalCell disruption goalvObjective function (monetary（金錢） value of v product)vSmall scale cell disruptionv(1) ultraso

10、nicv(2) freeze thawing (熔化）v(3) force cells through 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 （氣穴）v(3) impingement (沖擊）vConditionv(1) conc

11、entration (4-175 g dry wt/liter)v(2) pressure (30-95 MPa)v(3) passes 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) reproducible

12、v(4) better design and optimize equipmentv(5) Turbulent eddies (湍流渦旋） Responsible 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

13、 glucan netv(3) source（酶的來源） and culture system kinetic analysisvA understanding the systemvB designing the small and large scale systems vC cytophaga （吞噬細胞）system exhibits high protease activity, higher conversion and higher lysisrate at longer timevD oerskovia systems high glucanase low protease a

14、ctivity, contain some inhibitorsvE an initial lag in yeast lysis by both enzymevF simple two 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

15、 淀粉酶vModulate 調節(jié)vInterferons 干擾素vViscous fluids 粘性流體vSignal peptide 信號肽vCarboxypeptidase 羧肽酶vAlkaline phosphatase 堿性磷酸酯酶vGlyceraldehyde-3-phosphate dehydrogenase 甘油醛-3-磷酸脫氫酶vReduced nicotinamide adenine 還原型尼克酰胺腺嘌呤vDinucleotide 二核苷vReductase 還原酶vHydrophobic 疏水的vHydrophilic 親水的New words and vocabulari

16、es (2)vPathogens 病源體vBall mills 球磨機vResonance 共鳴，回聲vVibration 振動， 擺動vDissipate 分散vPinpointing 最佳點,針尖vOscillate 振蕩vCandida utilis 假絲酵母vExtrusion 噴出vSapphire 蘭寶石vJewel 寶石vThermocouple 熱電偶vSleeve 袖子，套vHydrodynamic 流體力學vHydroxylase enzyme 羥化酶vSynergistic 增效，協(xié)同作用vGlucan 葡聚糖vMorphology III initial fractio

17、nationvPrimary isolation stepv(1) adsorptionv(2) extractionv(3) precipitationv(4) distillationv(5) filtration v(6) solvent evaporationvRequirementvSignificant volume reductionInterest product in solutionv(1) gradually remove impuritiesv(2) increase product concentrationv(3) chose methods depends on

18、scale and organismv(4) washing stages followed by dewatering stepvYeasts easy to deal withv(1) 10 m in diameter 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) min

19、imize degradation cell wall following rupturev(2) minimize cell fragment proportionv(3) restrict viscosity increasev(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) adsorpti

20、onv(2) precipitationv(3) membrane separation v A cross flow filtrationv B microfiltrationv C ultrafiltrationv(4) liquid-liquid 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 con

21、centration of biological productv(6) cell particles and nucleic acids removedv(7) large volume reductionA adsorptionv(1) purify proteinv(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 adso

22、rbentv(5) expanded bedv(6) fluidized bedExpanded bed adsorptionv(1) eliminate a step for removal of cell or debrisv(2) scale up simple (similar to packed bed)v(3) combine clarification, concentration and purificationv(4) could be used for viscose fluidv(5) high biomass content of feedstockv(6) expan

23、ded bed stabilized by wide distribution of particle sizeNew words and vocabularyvColloidal （膠狀的）vGlycan （聚糖）vLysate （溶解物）vAerosol（懸浮微粒,氣溶膠）vParticulate （微粒）vFluidized bed （沸騰床）vStreptomycin （鏈霉素）vNovobiocin （新生霉素）vAnnexin V （膜聯(lián)蛋白一類被鈣離子活化后可與膜磷脂結合的蛋白）Scout （偵察）vWashing （洗滌）vElution （洗脫）New words and v

24、ocabularyvReverse phase high pressure liquid phase chromatography (反相HPLC）vCircumvented （包圍）vHybridoma （雜合細胞，(淋巴細胞)雜交瘤）vPenicillin （青霉素）vTetracycline （四環(huán)素）vCephalosporin C （頭孢菌素C）vDivinyl benzene （二乙烯苯）vStyrene （苯乙烯）B membrane separationvFunction : Clarification and sterilizing vProblems v(1) Solid-

25、liquid interface adsorptionv(2) significant surface area (500cm2)vMembrane materials v(1) hydroxyl-modified hydrophilic polyamide v(2) hydrophilic polyvinylidene fluoride (PVDF) 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

26、(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 inactivationv(2) chemical treatmentv(3) denaturant (urea)v(4) -propiolactone（丙內酯）-UV light exposurev(5) membrane f

27、iltrationv 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）vScale-down （按比例縮減）vDiscernible（可辯別的）vSteady state（穩(wěn)態(tài)）vAsymptotically （漸近的）vLow-binding protein membranev

28、-propiolactone UV light （-丙內酯-紫外光）vMonotonically（無變化的，單調的）vPlatelet （血小板）vImmunodiseasevHydraulic permeability （水滲透性）vFiltration resistance（過濾阻力）vPure water permeability (PWP) 純水透過性Vocabularies Domain disulfide bonds, signal peptide, turbulent eddies Aqueous two-phase partitioning expanded bed adsor

29、ption 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 Which differences between Mammalian cell proteins v and microorganism proteins ?v3 Which methods

30、 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 in protein solution ?Chapter 2 SecC UltrafiltrationvExample： antibodies productionv(1) ant

31、ibodies 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 frame ultrafiltration system v D gel polarization （濃差極化） reducedv F acceptable fl

32、ux 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 and vocabularyMandate （命令，要求）Diagnostics 診斷學Cytokines 細胞因子Blood clotting f

33、actors （凝血因子）Tangential plate 切向板，錯流過濾板Undermine（破壞）Integrity （完整）Tryptic -casein peptides -酪蛋白肽Antihypertensive （抗高血壓的）Immunomodulatory 免疫調節(jié)劑Plasmin 血纖維蛋白溶解酶Transmission 輸送Detrimental 有害的Interfacial tension 表面張力Emulsion liquid membrane 乳狀液膜Carrier 載體Diffusivity 擴散率D cross-flow filtrationvAffinity c

34、ross-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 incorporate ligandsv(3) high protein loss in washing stepNew words an

35、d vocabulariesvAffinity cross-flow filtrationvEscort 簇vSupernatant 上清液vMonomolecular layervInnermost 內心的，最里面的vLangmiur isothermvGlutaradehydevSchiff basesvSodium borohydridevMicroparticles E Emulsion liquid membrane vPenicillin G extraction by emulsion liquid membranevApplicationvConcentrate weak ac

36、ids, 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) acidification at pH 2v(3) neutralization with calcium carbonatev

37、(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 different subprocessesv(3) scale level of operationNew

38、 words and vocabulariesvEmulsion liquid membrane 乳化液膜vAmine 胺vCarrier 載體vPancreas 胰腺vHyperglycemia 高血糖vDiabetes mellitus 糖尿病vPorcine origin 豬器官vIsoelectric point 等電點vReverse-phase chromatography 反相層析vScarce 缺乏的，不足的vProprietary 所有權G precipitation vApplying high resolution technique at earlier stage v

39、(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 Precipitation factorsv(1) salt and cosolvents (

40、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 separating lactalbumin(-1A

41、)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 molecule and facilita

42、te 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, polyligands and liga

43、nd 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 concentr

44、ationSTI 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 large

45、 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

46、(93.3%, 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 親和沉淀vHeterobifunctional ligand 異種雙功能試劑vChitosan 殼聚糖vOligomeric 寡聚體的vSoybean trypsin inhibitor 大豆蛋白酶抑制劑vChymot

47、rypsin 胰凝乳蛋白酶，糜蛋白酶vSupersaturation 過飽和v-lactalbumin 乳白蛋白vWhey protein 乳清蛋白vMammary gland 乳腺vTrytophan 脯氨酸vTransitions 過渡vTurbidity 混濁vSurfactant 表面活性劑vPhospholipids 磷脂vAvidin 生物素結合蛋白vBiotin 生物素vDimyristoylphosphatidylethanolamine 二肉豆蔻酰磷酸乙醇胺vOctaethylene glycol mono-n-dodecyl-ether 八乙烯醇單十二烷基醚vHastene

48、d 促進，加速vPolymerized liposome 聚合脂質體vRedispers 再分散vSteric hindrance 空間位阻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) Tw

49、o ion exchange 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) prote

50、in content from 11 to1.5mg/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 us

51、ing HPLCv(2) linear adsorption 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

52、素vPharmacia columnvDose 劑量vPyrogen 熱原vMiniscule 小字的vDivinylbenzene polystyrene copolymer 苯乙烯-二乙烯苯共聚物vAspartame 門冬氨酰苯丙氨酸甲酯（比蔗糖甜200倍的甜味劑）vPhenylalanine 苯丙氨酸vAspartic acid 天門冬氨酸vAsparagine 天門冬素，天冬酰胺酸vAdsorption isotherm 吸附等溫線vHomologues 類似物，同族體IV high-resolution fractionationvPrevious purification step

53、s v(1) volume reductionv(2) clarification v(3) adjustment of crude extractvChromatography stepsv(1) cation exchangev(2) anion exchangev(3) affinity chromatographyv(4) hydrophobic chromatographyA chromatography proceduresvAffinity chromatographyv(1) nonspecific adsorptionv(2) expensive labile ligandv

54、(3) difficulties of adsorbent sterilizationv(4) biomimetic ligandv(5) natural ligand expensive and decreased activity when v immobilized.v(6) pseudo-ligandv(7) ligand leakage and product contaminationvLarge scale purification v(1) remove low levels of impuritiesv(2) reduce pyrogenv(3) exchange into

55、buffer systems suitable for formulationv(4) gel filtration chromatography has low productivityvSEB:Staphylococcal enterotoxin 葡萄球菌外毒素Final products analysis v(1) SDS-PAGE (single band)v(2) gel filtration (single peak)vDeveloping large-scale purification stepv(1) new mediav(2) equipmentv(3) optimizat

56、ionUltrafast HPLCv(1) instrumentation v A selectivityv B sensitivityv(2) resinv A rapid association and dissociationv B material geometry play significant rolev(3) obtain quantitative product recovery estimationv(4) visualize wide variety of host cell impuritiesv Posttranslational modification ( dea

57、midation, proteolysis v and glycosation)v(5) rapid HPLC significancev A monitoring processv B optimization of protein purity v C developing large scale processPurification and characterization of lamb pregastric lipasev(1) play an important role in fat digestion in newborn animalsv(2) enhance cheese

58、 food flavorsv(3) initial purification stepsv A acid precipitationv B pepsin digestionv C Econo-Pac blue affinity chromatography v(4) polishing stepv A enhance purityv B removal of pyrogenv C enhance specific activityv(5) chromatography media v A laboratory scale, pilot plant and production level ha

59、ve different v requirementv B no lot-to-lot consistency.v C efficiency (cost effectiveness) and quality compromisev D cost media, purification step number, and fastest throughput New words and vocabulariesvTrain 行列vPick-up 提取vBiomimetic 仿生vPseudo ligand 假配基vBuffer systems 緩沖體系vUltrafast 超快的vAssociat

60、ion 結交，結合vDissociation 解離vChimeric 妄想的，vGlycosation 糖基化vParamount 極為重要的vHeterogeneities 異種性vLamb pregastric lipase 小羊胃前脂肪酶vPepsin 胃蛋白酶vFlavors 香料vEcono-pac Blue affinity chromatographyvBring out 顯示出vLot-to-lot 批-批vAlbumin 清蛋白，白蛋白V Conclusionsv(1) Three stages of bioseparations are v interdependence