生物分离工程双语版3

1、I Introduction v(1) appropriate sequencev(2) utilization (therapeutic, diagnostic)v(3) operation scalev(4) role of final fractionation v A impuritiesv B saltsv C pathogensv D endotoxinsv E contaminating proteins v(5) low volume and expensive proceduresv(6) special attentionv A amount of product dena

2、turationv B quality II Chromatography procedures vA gel filtration chromatographyvB Hydroxylapatite chromatographyvC Hydrophobic interaction v chromatographyvD Affinity chromatographyvE Ion-Exchange ChromatographyA gel filtration chromatographyv(1) mechanism v A molecular sizev B mobile solvent phas

3、ev C stationary phasev(2) Rigidity of beads in large scale operation v(3) traditional gel filtration chromatography mediav A Sephadexv B Superosev C Sephacryl v D Trisacrylv E Biogel Pv F Cellulofinev G Fractogelv(4) low product volume suitable for gel filtration (5) activity loss A final fractionat

4、ion B previous bioseparation stepsExmaple 3.1 carboxylesterase purification v(1) Material ：Bacillus stearothermophilus噬热脂杆菌v(2) Step utilized v A centrifugationv B DEAE-Sephacel ion-exchange chromatographyv C gel filtration chromatographyv(3) Result analysis v A single low-mobility band (PAGE)v B Tw

5、o bands of higher mobility (after precipitation and anion v exchange） v C just single band of high mobility (further gel filtration) v D gel filtration and SDS-PAGE indicate esterase is v monomeric protein with MW 40000 Exmaple 3.1 carboxylesterase purification v(4) Reason v A active monomer associa

6、ting multiple formsv B multiple form have different chromatography and v electrophoretic properties v C reaction equilibrium affected by pH, salt v concentration, enzyme dilutionv D monomers or multimeric enzyme forms affect v activity and stabilityDifference between crude and purified esterase form

7、sv(1) Thiol-containing compounds in crude v material lead to cysteine-containing protein v denaturation v(2) Proteolysis activity in crude materialv(3) Coprecipitation with less stable compounds v in crude materialv(4) Conformational changes in processing v stepsResults and discussion about table 3.

8、1 v(1) 48% recovery is high yield (not so high v purification factor)v(2) State change of enzyme aggregation lead v to activity lossv(3) Analyze cause permit minimize activity loss Purification of antigenized immunoglobulins with monomethoxypolyethylene glycol (1) polyethylene glycol (PEG) usage v A

9、 nontoxic v B nonimmunogenic v C internal （内用）use in humansv D PEGylated proteins preserve biological activity (2) various degree of derivatization occur A protein microheterogeneity B distribution of number and portion of PEG C PEG polydispersity (3) Chemical reaction mPEG attached to AIgs Purifica

10、tion of antigenized immunoglobulins with monomethoxypolyethylene glycol(4) Purification result of AIg-mPEG using size exclusion chromatography A ammonium hydrogen carbonate as buffer system B Ultrogel AcA-44 gel column C two elution peaks including AIgs-mPEGs and unconjugated AIgs, and mPEG (5) Furt

11、her fractionating first peak using Q300 anion-exchange column A first peak with high PEGylated Ig-Hemaglutinin(HA) B second peak containing mildly PEGylated Ig-hemaglutinin C third peak unconjugated control Ig-HA(6) 6-8% degree of derivatization and long half life Example 3.2 Tissue plasminogen acti

12、vator purification v(1) material： animal cell and bacterialv(2) tPA 2200 USD/dose 20 times than streptokinase v(3) streptokinase use only once, tPA more than oncev(4) tPA model productv A flagship product for biotechnolotyv B first product for market using geneticallly v engineered mammalian cells i

13、nstead of v recombinant bacteriav(5) market requirement with 11000 g tPA/YeartPA purification protocolsv(1) Affinity chromatographyv(2) Ion-exchange chromatographyv(3) Amino acid Sepharose v(4) Gel chromatographyv(5) Ultrafiltrationv(6) Centrifugationv(6) Microfiltrationv(7) Solubilizationv(8) Cleav

14、agev(9) refoldingt-PA from CHO and E.coli(1) 5 steps purification for CHO and 16 steps E.coli.(2) Affinity chromatography and gel chromatography used in the end of purification process to remove impurities and salts (3) t-PA purity greater than 99.5% (4) Clearance of endotoxin with 99.999%(5) Drop i

15、n product yield ( refolding 20%, ultrafiltration 56%Xylanase purification v(1) material: thermophilic ascomycete(子囊菌)v(2) xylans (major hemicellulose in angiosperms)v(3) three separation stepsv A ultrafiltration stepv B anion exchange step(Q-sepharose column)v C gel filtration (superose 12 column)v(

16、4) xylanase interact with gel filtration to result v in conformational change (high tyrosine v content)Purification of two isoenzymes of xylanase from streptomycesvPurification sequencev(1) ammonium sulfatev(2) precipitationv(3) centrifugationv(4) desalting (Sephadex G 25)v(5) DEAE-Sepharose FF colu

17、mnv(6) CM-Sepharose columnv(7) concentration by ultrafiltrationv(8) gel filtration on Sephadex G 75 columnv(9) freeze-dry Purification of highly thermostable glucose isomerase (1) material : thermohilic bacterium Thermotoga maritima （2）55% fructose produced at 95-100C（3）purification step A centrifug

18、ation B Q-Sepharose column C Phenyl-650 M hydrophobic resin column D Q-Sepharose HP anion exchange E Hiload superdex 200 gel filtration chromatography (4) SDS-PAGE A Single band B With molecular weight 45000 C Homotetrameric structure D Most common oligomeric state of glycose isomeraseExample 3.3 pu

19、rification of two endo-glucanase from aerobic fungusv(1) material : aerobic fungus penicillium capslatum v(2) glycans(聚糖） problems in beer industryv A precipitatev B hazes （混浊）v C gel in stored beerv D gumminessv(3) Purification stepv A freeze-dry of crude extractv B gel filtration on a Sephacryl S-

20、300 columnv C ion exchange on a DE-52 cellulose v D ultrafiltrationv E electrophoresisv(4) endo-glucanase A and endo-glucanase Bv(5) gel filtration used earlier v(6) crude extract is more stable and the purified enzyme.Purification protocol for -glucosidase from clostridium thermocellum v(1) materia

21、l: clostridium(梭菌)therocellum v(2) degradation of crystalline cellulose produce cellobiose v by cellulase complex v(3) -glucosidase metabolize cellobiose v(4) purification stepsv A sonicationv B centrifugationv C DEAE-Sepharose CL-6B chromatographyv D ion exchange chromatographyv E high performance

22、Nono Q HR columnv F ultrafiltrationv G chromatofocusing on Mono P HR columnv H gel filtrationv(5) homogeneous enzyme obtainedPectin methylesterase purificationv(1) material: bacillus subtilisv(2) pectin is major structural component in plant cell wallsv(3) pectinase degrade pectin v(4) PME usagev A

23、clarification of cider v B deesterification of pectin to methanol and pectatev(5) purification stepsv A centrifugationv B ultrafiltrationv C two gel filtration v(6) homogeneous PME obtainedv(7) protein loss by leakage in UF membrane (PME 36000 Da, molecular v cutoff 10000Da)v(8) low activity in abse

24、nce of salts (nonspecific binding or aggregationHydroxyapatite chromatographyv羟基磷辚石(, Ca10(PO4)6(OH)2) v（1）纯化蛋白质的无机介质。v（2）廉价，易于大规模应用，v（3）使用后易于清洁 v吸附机理与规律：偶极离子作用而不是离子交换作用v(1) 每一个正电荷区周围都有负电荷区，反之v 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v吸附：v(1) pH在69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

25、中进行（3）盐可以采 用恒定或梯度形式应用。B Hydroxylapatite chromatographyvExample 3.5 purification of clostridium thermoceilum -v glucosidase Bv-glucosidase B usagev Catalyze hydrolysis of -glucoside linkages between v glucose and alkyl, aryl, or saccharide groupvBioseparation precedures vA ammonium sulfate precipitat

26、ionvB Q-sepharose FF columnvC butyl-sepharosevD hydroxylapatitevPurification resultvA table 3.9vB stability increased by addition of divalent cation at 45-60CChromatography media vSilica gel vA regular shape (sphere)vB uniform size (3-10m)vC uncoat surface dissolved by alkaline solutionvD alkyl grou

27、ps tend to be cleaved in acidic v solution vOther material vA titania vB zirconia 硅胶v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v（1）化学组成 SiO2.XH20，v（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。v（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v 面。v硅胶的分类（常以孔径大小来分），v(1) 特细孔硅胶 (0.8nm以下)。 (2) 细孔硅胶（1.52.0nm )v(3) 中孔硅胶 (4.0一5.0nm)v(4) 粗

28、孔硅胶 (10.nm以上)v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Sorbitol and sorbitol dehydrogenasevSorbitol application: sweetener and food additivevSorbitol dehydrogenase application ( oxidizes sorbitol to D-fructose )vSorbitol dehydrogenase purification vA (NH4)2SO4 precipitationvB Q-Sepha

29、rose Chromatography vC procion-blue affinity chromatographyvD hydroxylapatite chromatographyvE ultrafiltrationvPurificaton result vA table 3.10vB single protein band by SDS-PAGEvC purified enzyme quite stable and stability increase by addition v of sucrosePurification of D-xylulokinase vMaterial: ye

30、ast pichia(毕赤酵母属)stipitis vD-xylose : The second largest amount of sugar is naturally availablevD-xylullokinase（木酮糖激酶） convert D-xylose to ethanolvPurification protocol:vTwo steps hydroxylapatite chromatographyvUltrafiltrationvPurification resultvA table 3.11vB homogeneity samplevC major activity lo

31、ss occur in the first adsorption columnvD partially purified enzyme unstable at 4 C , but stable at -20CvE all kinase reaction require divalent metal ion (Mg 2+) New words and vocabulariesvChitanase 甲壳素酶vConfectioning 调制部分,调糖膏）vEntail 使必须，使承担vRigidity 刚性vCarboxylesterase 羧酸酯酶vEsterase 酯酶vMobility 迁移

32、率vStatus 状况vMultimeric 多聚体的New words and vocabulariesvThiol 硫醇v cysteine 半胱氨酸vUnderscore 划线，强调vMonomethoxypolyethylene glycol vMicroheterogeneity 微观异种性vPolydispersity 多分散性vHemaglutinin 凝集素vConjugation 结合物vHalf life 半衰期vTissue plasminogen activator 组织纤维蛋白 酶原激 活剂 vStreptokinase 链激酶New words and vocabu

33、laries vBovine growth hormone 牛生长激素vChinese hamster ovary (CHO) 中国鼠卵细胞vCleavage 裂解，分开vXylanase 木聚糖酶vAscomycete 子囊菌属vHemicellulose 半纤维素vAngiosperm 被子植物vLignin 木质素vTyrosine 酪氨酸vXylanhydrolase 木聚糖水解酶vActinomycetes 放线菌New words and vocabularies vSweetener 甜味剂vFructose 果糖vHigh fructose corn syrup 高果糖浆vHe

34、motetrameric 均一的四聚体vHaze 混浊vGumminess 树胶，粘性vPectin 胶质vPectinase 胶质酶vTexture 结构，质地vCider 苹果酒vFlagellin 鞭毛蛋白vHydroxylapatite 羟基磷灰石Questions v1 What is mechanism of gel filtration ?v2 Which differences between crude proteins and v purified proteinsv3 Why some enzymes were modified with v mPEG?v4 Why st

35、reptokinase used only once, while tPA v used more than once?v5 What is mechanism of hydroxylapatite ?v6 Key words: polydispersity, hemaglutinin, v Chinese Hamster ovary(CHO), high fructose v corn syrup, hydroxylapatite. Chapter 3 2stHydrophobic chromatographyvExample 3.7 purification of feruloy/p-co

36、umaroyl esterase vMaterial: fungus penicillium pinophiliumvXylans : major constituents of woods and agricultural residuesvP-coumaric esterase: esterifies the L-arabinose residues in the v xylan backbone (important modificationvPurification procedures:vA Amicon ultrafiltrationvB DEAE-Sepharose CL-6B

37、anion-exchange chromatographyvC hydrophobic interaction columnvPurification resultvA table 3.12vB Single band by SDS-PAGE and 57000 Daton MWvC stabile from 37-55C疏水层析v 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 v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 v介质配基密度:4080mol/ml胶, v吸附容量：牛血清蛋白(BSA)

38、大于40mg/ml疏水层析介质制备过程示意图 工艺条件v吸附：v(1) 盐浓度：高盐， 12M (NH4)2SO4 或NaClv(2) pH: 中性或偏酸性v淋洗：0.52%表面活性剂v洗脱:v(1) 低盐，0.10.5M NaCl, 再配合pH变化。v(2) 低浓度促溶剂,0.10.5 %NaSCN, 或2040%乙二醇v疏水层析实验方案：New words and vocabularies vHydroxylapatite 羟基磷灰石vAlkyl 烷基vAryl 芳基vSaccharide 糖类vButyl-Shpharose 丁基-SepharosevSilica gel 硅胶vUnco

39、ated 未涂层vTitania 二氧化钛vZirconia 氧化锆 vSorbitol 山梨醇vD-glucitol 葡萄糖醇vAcyclic polyol 无环多醇vNicotinamide adenine dinucleotide (NAD) 尼克酰胺腺嘌呤二核苷酸vXylitol 木糖醇vSucrose 蔗糖vD-xylulokinase 木酮糖激酶vPentose 戊糖vPhosphorylation 磷酸化vFeruloyl / p-coumaroyl esterase （氯苯二酚氨醇香豆素脂酶）vArabinose 树胶醛糖，阿拉伯糖Affinity chromatography

40、 vExample 3.8 purification of chitanase vMaterial: trichoderma （木霉）barzianumvPurification protocolvA ammonium sulfate precipitationvB Q-Sepharose ion-exchange chromatographyvC Sephadex G-100 gel filtrationvD phenyl-Sepharose CL-4B hydrophobic interaction v chromatographyvPurification resultv table 3

41、.13Cluster model of multivalent affinityvSelectivity is dependent on ligand and matrixvMultivalent interactionvA two or more ligand in a cluster binding two or v more contactsvB cooperativity due to proximity and entropic vC association constants for ligand pairs 100-fold v greater than constant for

42、 single ligand, while model v constant should be seven orders of magnitude v highervD geometrical-steric constraint 亲和层析亲和层析v亲和层析(Affinity Chromatography)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。v生物体内相互作用的分子对：v(1) 酶底物或抑制剂v(2) 抗原抗体。v(3) 激素受体。v(4) 糖蛋白与凝集素,v(5) 生物素生物素结合蛋白等 基质活化方法与配基偶联基质活化方法与配基偶联 v活化(activation):

43、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。v(1) 大分子配基如蛋白质等可与活化基质直接v 偶联。v(2) 小分子配基, 在基质与配基之间插入若干v 碳原子手臂(spacer),然后再与配基偶联。v(3) 环氧氯丙烷, 1,4丁二醇缩水甘油醚本身v 是活化剂亦具有手臂作用。活化方法（1）vA 溴化氰法溴化氰法v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v优点优点:（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）

44、偶联条件温和,特别适用于偶联敏感性生物大分子。v缺点：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）vB 环氧氯丙烷活化环氧氯丙烷活化 v（1）所形成的共价键稳定,配基不易落脱v（2）自动引入手臂,v（3）有更小的非特异性吸附,v（4）活化操作简单易行,危险性相对较小v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-exchange chromatographyvExample 3.10 purificati

45、on of k-carrageenasevMaterial: pseudomonas （假单孢菌属）carrageenovora vCarrageenans（角叉菜聚糖）: major component of cell wall structure in red algaevCell wall degrading enzyme : used for obtaining protoplasts and v understanding structure of carrageenansvPurification protocolvSmall scalev A (NH4)2SO4 precipit

46、ationv B Sephadex column gel filtrationv C CM-sepharose CL-6B columnvLarge scalev A ultrafiltrationv B S-Sepharose FF cation exchange vPurification result v A table 3.14 v B double band (32000 and 34000 Da)离子交换法v概述：利用离子交换树脂分离生物物质的方法v特点：v（1) 浓缩效应v (2) 成本低，设备简单，操作方便v (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

47、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，v-PO（OH）2v -PHO（OH）vRSO3H+NaCl RSO3Na + HClv交换能力与pH无关弱酸性阳树脂v-COOH，v-OH (酚羟基）v强碱性阴树脂弱碱性阴树脂Example 3.11 Production of blood protein using ion-exchange vMaterial: human placental blood and human plasmavMatrix : silica particlesv A rigidityv B incompressibilityv C low pre

48、ssure dropv D large pore diameterv E coated by suitable polymers such as polysaccharidev F possible application on an industrial scalevResults and discussion v A Production scale 20000 liters / day and 140 Kg of proteinv B Sucrose and albumin minimize denaturation of blood coagulating factorv C Exte

49、nsive dialysis or diafiltration lead to denaturation of proteins when v removal of competitive ligandsv D Mild elution conditions with metal chelating agents minimize metal v leakage离子交换提取蛋白质v传统离子交换剂不适用于提取蛋白质v（1） 交联度大 （大分子不能进入）v（2） 电荷密度高(结合太强）v（3） 骨架憎水性强（蛋白质易变性)v亲水性离子交换剂v（1）亲水性大v（2） 孔径大v（3） 体积随pH 及离

50、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v亲水性离子交换容量不能达到无机离子交换容量v（1）蛋白质不能进入活性中心v（2） 一个蛋白质与多个功能基发生作用吸附机理v （1）静电吸力v （2）憎水v （3）氢键v （4）被吸附蛋白表面进一步吸附蛋白质的离子交换层析v层析介质：DEAE-纤维素，CM-纤维素v吸附条件：v (1) 缓冲液pHv (2) 蛋白浓度控制 （5mg/ml)v（3）离子强度v (4) 道南效应(Donnan effect) 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2

51、0-40cm, 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；c pH 变化大 New words and vocabulariesvAcetamido 乙酰氨基vEndoglycosidase 葡萄糖内酯酶vPhenyl-Sepharose CL 4B 苯基-vP-nitrophenyl-N-actylglucosamine 对硝基苯-N-乙酰葡萄糖胺vEntropic 熵vRabbit muscle dehydrogenase 兔肌脱氢酶vClusters 簇vLactate dehydrogenase 乳酸脱氢酶vCibacron blu

52、e cellulosevOrders of magnitude 数量级New words and vocabulariesvRandom 随机vK-carrageenase k-角叉菜酶v algae 海藻vSeaweed 海草，海藻vProtoplasts 原生质体vCM-Sepharose CL-6BvPhycocolloid 藻胶vPlacental 胎盘的vChloroform 氯仿vHemoglobin 血红素vElectrolytes 电解质III Crystallization vCrystallization of bacterial luciferase（萤光素酶）vMate

53、rial: marine bioluminescent（生物发光） bacteriumvCrystallization stepsv (1) microfiltrationv (2) sitting-drop vapor diffusion method with v crystallization platesv(3) Biomek 1000 automated laboratory system v (accuracy , reproducibility, and precision)Example 3.12 purification and crystallization of lipa

54、se vMaterial: geotrichum（地丝霉属） candidum（念珠菌）vLipase have a high specificity for fatty acids having at least one v cis-9 double bond.vPurification stepsv(1) Q-Sepharose columnv(2) Phenyl-Sepharose columnvCrystallization v(1) hanging-drop method using multichamber platesv(2) crystallized in the presen

55、ce of PEGv(3) crystals size depends on molecular weight of crystallization v agentv(4) five different crystals obtained depending on the amount of v glycosylation v(5) pure enough to obtain X-ray diffraction dataPurification and crystallization of lipase vPurification Protocolv(1) ion-exchange chrom

56、atographyv(2) S-Sepharose fast flowv(3) Phenyl-Sepharose CL-4B hydrophobic interaction v chromatographyv(4) hydroxylapatite adsorption chromatographyv(5) -aminohexyl agarose affinity chromatographyvCrystallization v(1) bundles of needles obtained in ammonium sulfitev(2) rhombic(斜方） crystals obtained

57、 in ammonium sulfatev(3) pure crystal were suitable for X-ray diffraction studiesAntibiotics crystallizationvExample 3.14 penicillin crystallizationvBasic bioseparation protocolv(1) filtrationv(2) centrifugationv(3) liquid-liquid extractionv(4) crystallization vDiscussionv(1) purification of antibio

58、tics under sterile conditionv(2) rapid processingv(3) sensitive to temperaturev(4) extra care to prevent contamination or degradation (-v lactamase-producing organisms)vExample 3.15 purification and crystallization of cephalosporinAntibiotics crystallizationvBioseparation protocolv(1) solvent extrac

59、tionv(2) ion-exchange resinv(3) salting-out v(4) activated carbon columnv(5) precipitationv(6) crystallizationv A potassium or sodium salt of cephalosporinv B miscible solventv C Coper, nickel, or lead salt crystallization from v aqueouss solutions IV other techniques vExample 3.16 purification of -

60、galactosidasevMaterial: Aspergillus （曲霉）fonsecaeusv-galactosidase : hydrolysis of lactose in milk and whey in dairy industryvProtocol v(1) ultrafiltraction-diafiltrationv(2) isopropanol precipitation vPurification result v(1) table 3.18 v(2) isopropanol precipitation as high-resolution fractionation