生物分離工程-雙語版-3教學內(nèi)容

1、生物分離工程-雙語版-3II 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 phasev C sta

2、tionary 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 fractionation B pr

3、evious bioseparation stepsExmaple 3.1 carboxylesterase purification v(1) Material ：Bacillus stearothermophilus噬熱脂桿菌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 Two bands

4、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 associating mul

5、tiple 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 formsv(1) Th

6、iol-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 3.1 v(1) 4

7、8% 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 nontoxi

8、c v B nonimmunogenic v C internal （內(nèi)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 Purification of

9、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) Further frac

10、tionating 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 activator pu

11、rification 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 instead o

12、f 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) Cleavagev(9)

13、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 in produc

14、t 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(4) xylan

15、ase 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 columnv(6) C

16、M-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 centrifugation B

17、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 purificati

18、on 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 （混濁）v C gel in stored beerv D gumminessv(3) Purification stepv A freeze-dry of crude extractv B gel filtration on a Sephacryl S-300 colu

19、mnv 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) material: clost

20、ridium(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 Nono Q H

21、R 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 clarific

22、ation 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 absence of s

23、alts (nonspecific binding or aggregationHydroxyapatite chromatographyv羥基磷轔石(, Ca10(PO4)6(OH)2) v（1）純化蛋白質(zhì)的無機介質(zhì)。v（2）廉價，易于大規(guī)模應用，v（3）使用后易于清潔 v吸附機理與規(guī)律：偶極離子作用而不是離子交換作用v(1) 每一個正電荷區(qū)周圍都有負電荷區(qū)，反之v 亦然 . （2）蛋白質(zhì)在中性pH范圍具有最大的形成毗鄰 正負離子的可能性。工藝條件v吸附：v(1) pH在69之間 （2）低濃度緩沖液離子（通常是磷酸鹽） （3）低鹽濃度 洗脫：（1）增加緩沖液濃度（2）高鹽濃度中進行（3）鹽可

24、以采 用恒定或梯度形式應用。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 precipitationvB Q-

25、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 groups tend

26、to be cleaved in acidic v solution vOther material vA titania vB zirconia 硅膠v硅膠是由多聚硅酸經(jīng)分子間脫水而形成的一種多孔性物質(zhì)v（1）化學組成 SiO2.XH20，v（2）屬于無定形結(jié)構(gòu)，基本結(jié)構(gòu)質(zhì)點為Si一O四面體，相互堆 積形成硅膠的骨架。質(zhì)點間的空間即為硅膠的孔隙。v（3）硅膠中的水以羥基的形式和硅原子相連而覆蓋于硅膠表v 面。v硅膠的分類（常以孔徑大小來分），v(1) 特細孔硅膠 (0.8nm以下)。 (2) 細孔硅膠（1.52.0nm )v(3) 中孔硅膠 (4.0一5.0nm)v(4) 粗孔硅膠 (10.

27、nm以上)v應用：吸附層析劑與分配層析劑 優(yōu)先吸附極性分子及不飽和的碳氫化合物，用于天然產(chǎn)物分離， 可用在水溶液中或有機溶劑中Sorbitol and sorbitol dehydrogenasevSorbitol application: sweetener and food additivevSorbitol dehydrogenase application ( oxidizes sorbitol to D-fructose )vSorbitol dehydrogenase purification vA (NH4)2SO4 precipitationvB Q-Sepharose Chr

28、omatography 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: yeast pich

29、ia(畢赤酵母屬)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 loss occur

30、 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 甲殼素酶vConfectioning 調(diào)制部分,調(diào)糖膏）vEntail 使必須，使承擔vRigidity 剛性vCarboxylesterase 羧酸酯酶vEsterase 酯酶vMobility 遷移率vStatus

31、 狀況vMultimeric 多聚體的New words and vocabulariesvThiol 硫醇v cysteine 半胱氨酸vUnderscore 劃線，強調(diào)vMonomethoxypolyethylene glycol vMicroheterogeneity 微觀異種性vPolydispersity 多分散性vHemaglutinin 凝集素vConjugation 結(jié)合物vHalf life 半衰期vTissue plasminogen activator 組織纖維蛋白 酶原激 活劑 vStreptokinase 鏈激酶New words and vocabularies v

32、Bovine growth hormone 牛生長激素vChinese hamster ovary (CHO) 中國鼠卵細胞vCleavage 裂解，分開vXylanase 木聚糖酶vAscomycete 子囊菌屬vHemicellulose 半纖維素vAngiosperm 被子植物vLignin 木質(zhì)素vTyrosine 酪氨酸vXylanhydrolase 木聚糖水解酶vActinomycetes 放線菌New words and vocabularies vSweetener 甜味劑vFructose 果糖vHigh fructose corn syrup 高果糖漿vHemotetram

33、eric 均一的四聚體vHaze 混濁vGumminess 樹膠，粘性vPectin 膠質(zhì)vPectinase 膠質(zhì)酶vTexture 結(jié)構(gòu)，質(zhì)地vCider 蘋果酒vFlagellin 鞭毛蛋白vHydroxylapatite 羥基磷灰石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 streptokin

34、ase 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-coumaroyl

35、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 anion-ex

36、change chromatographyvC hydrophobic interaction columnvPurification resultvA table 3.12vB Single band by SDS-PAGE and 57000 Daton MWvC stabile from 37-55C疏水層析v 將疏水性基團如丁烷、辛烷、苯固定化到介質(zhì)上, 這些基團會與蛋白質(zhì)生物大分子上的疏水區(qū)親和 v介質(zhì)制備：瓊脂糖在有機溶劑中用CDI(羰基二咪唑)活化后再與芳胺或烷胺形成酰胺鍵得到疏水層析介質(zhì) v介質(zhì)配基密度:4080mol/ml膠, v吸附容量：牛血清蛋白(BSA)大于40mg/m

37、l疏水層析介質(zhì)制備過程示意圖 工藝條件v吸附：v(1) 鹽濃度：高鹽， 12M (NH4)2SO4 或NaClv(2) pH: 中性或偏酸性v淋洗：0.52%表面活性劑v洗脫:v(1) 低鹽，0.10.5M NaCl, 再配合pH變化。v(2) 低濃度促溶劑,0.10.5 %NaSCN, 或2040%乙二醇v疏水層析實驗方案：New words and vocabularies vHydroxylapatite 羥基磷灰石vAlkyl 烷基vAryl 芳基vSaccharide 糖類vButyl-Shpharose 丁基-SepharosevSilica gel 硅膠vUncoated 未涂層

38、vTitania 二氧化鈦vZirconia 氧化鋯 vSorbitol 山梨醇vD-glucitol 葡萄糖醇vAcyclic polyol 無環(huán)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 樹膠醛糖，阿拉伯糖Affinity chromatography vExampl

39、e 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 3.13Clust

40、er 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 single

41、ligand, while model v constant should be seven orders of magnitude v highervD geometrical-steric constraint 親和層析親和層析v親和層析(Affinity Chromatography)是利用生物體內(nèi)存在的特異性相互作用的分子對而設計的層析方法。v生物體內(nèi)相互作用的分子對：v(1) 酶底物或抑制劑v(2) 抗原抗體。v(3) 激素受體。v(4) 糖蛋白與凝集素,v(5) 生物素生物素結(jié)合蛋白等 基質(zhì)活化方法與配基偶聯(lián)基質(zhì)活化方法與配基偶聯(lián) v活化(activation): 基質(zhì)（matri

42、x）上的化學基團是不活潑的,無法與配基直接偶聯(lián),通過化學反應使介質(zhì)上化學基團處于活化狀態(tài)。v(1) 大分子配基如蛋白質(zhì)等可與活化基質(zhì)直接v 偶聯(lián)。v(2) 小分子配基, 在基質(zhì)與配基之間插入若干v 碳原子手臂(spacer),然后再與配基偶聯(lián)。v(3) 環(huán)氧氯丙烷, 1,4丁二醇縮水甘油醚本身v 是活化劑亦具有手臂作用?；罨椒ǎ?）vA 溴化氰法溴化氰法v溴化氰在堿性條件下與多糖上的羥基反應導入氰酯鍵或亞氨碳酸酯到基質(zhì)上,進而與配基偶聯(lián)v優(yōu)點優(yōu)點:（1） 適用于含羥基多糖及含羥基的合成基質(zhì)（2）用于含伯氨基小分子配基及伯氨基大分子配基的偶聯(lián)（3）操作步驟簡單,重現(xiàn)性好。（4）偶聯(lián)條件溫和,特

43、別適用于偶聯(lián)敏感性生物大分子。v缺點：缺點：（1）形成的異脲鍵易產(chǎn)生非特異性吸附,（2）共價鍵不穩(wěn)定,配基容易脫落,（3）活化操作危險性大，反應后的殘余液需經(jīng)處理再排放 溴化氰活化與配基偶聯(lián)化學反應式 活化方法（2）vB 環(huán)氧氯丙烷活化環(huán)氧氯丙烷活化 v（1）所形成的共價鍵穩(wěn)定,配基不易落脫v（2）自動引入手臂,v（3）有更小的非特異性吸附,v（4）活化操作簡單易行,危險性相對較小v缺點：在強堿性條件反應，不適用于堿敏感物質(zhì)。 環(huán)氧氯丙烷活化反應式 環(huán)氧基的氨化及偶聯(lián)配基反應 Ion-exchange chromatographyvExample 3.10 purification of k-

44、carrageenasevMaterial: pseudomonas （假單孢菌屬）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 precipitationv B

45、 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)離子交換法v概述：利用離子交換樹脂分離生物物質(zhì)的方法v特點：v（1) 濃縮效應v (2) 成本低，設備簡單，操作方便v (3) 生產(chǎn)同期長，pH 變化大離子交換樹脂：不溶于酸堿和有機溶劑的固態(tài)高 分子化合

46、物骨架活性離子強酸性陽離子交換樹脂 -SO3H，v-PO（OH）2v -PHO（OH）vRSO3H+NaCl RSO3Na + HClv交換能力與pH無關弱酸性陽樹脂v-COOH，v-OH (酚羥基）v強堿性陰樹脂弱堿性陰樹脂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 pressure dr

47、opv 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 Extensive di

48、alysis 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離子交換提取蛋白質(zhì)v傳統(tǒng)離子交換劑不適用于提取蛋白質(zhì)v（1） 交聯(lián)度大 （大分子不能進入）v（2） 電荷密度高(結(jié)合太強）v（3） 骨架憎水性強（蛋白質(zhì)易變性)v親水性離子交換劑v（1）親水性大v（2） 孔徑大v（3） 體積隨pH 及離子強度變化小親水

49、性離子交換劑功能團離子交換劑的交換容量v親水性離子交換容量不能達到無機離子交換容量v（1）蛋白質(zhì)不能進入活性中心v（2） 一個蛋白質(zhì)與多個功能基發(fā)生作用吸附機理v （1）靜電吸力v （2）憎水v （3）氫鍵v （4）被吸附蛋白表面進一步吸附蛋白質(zhì)的離子交換層析v層析介質(zhì)：DEAE-纖維素，CM-纖維素v吸附條件：v (1) 緩沖液pHv (2) 蛋白濃度控制 （5mg/ml)v（3）離子強度v (4) 道南效應(Donnan effect) 介質(zhì)內(nèi)外鹽濃度及 pH 不同 (5) 上樣量：1-5%BV，高徑比20:1, L： 100cm(恒定洗 膠）； 5-10%qm, 柱長度20-40cm,

50、高徑比5：1 （分步或梯度） 洗脫：恒定，分步，梯度pH 梯度：a 緩沖量大；b 離子強度變化；c pH 變化大 New words and vocabulariesvAcetamido 乙酰氨基vEndoglycosidase 葡萄糖內(nèi)酯酶vPhenyl-Sepharose CL 4B 苯基-vP-nitrophenyl-N-actylglucosamine 對硝基苯-N-乙酰葡萄糖胺vEntropic 熵vRabbit muscle dehydrogenase 兔肌脫氫酶vClusters 簇vLactate dehydrogenase 乳酸脫氫酶vCibacron blue cellul

51、osevOrders of magnitude 數(shù)量級New words and vocabulariesvRandom 隨機vK-carrageenase k-角叉菜酶v algae 海藻vSeaweed 海草，海藻vProtoplasts 原生質(zhì)體vCM-Sepharose CL-6BvPhycocolloid 藻膠vPlacental 胎盤的vChloroform 氯仿vHemoglobin 血紅素vElectrolytes 電解質(zhì)III Crystallization vCrystallization of bacterial luciferase（螢光素酶）vMaterial: ma

52、rine bioluminescent（生物發(fā)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 lipase vMate

53、rial: geotrichum（地絲霉屬） 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 presence of PE

54、Gv(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 chromatograph

55、yv(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 in ammo

56、nium 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 antibiotics und

57、er 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 extractionv(2)

58、 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 -galactos

59、idasevMaterial: 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 stepv(3

60、) diafiltration to remove major low molecular-weight contaminants v(4) further purification v A hydroxylapatite v B gel filtrationv C anion exchange (Fast phase liquid chromatography,FPLC)v(5) both partial purification and high purification providedExample 3.17 purification of -glucosidasevMaterial