生物分離工程雙語版3

Chapter3

1stHighresolutionfractionationprocessesIIntroduction

(1)appropriatesequence(2)utilization(therapeutic,diagnostic)(3)operationscale(4)roleoffinalfractionationAimpuritiesBsaltsCpathogensDendotoxinsEcontaminatingproteins(5)lowvolumeandexpensiveprocedures(6)specialattentionAamountofproductdenaturationBqualityIIChromatographyprocedures

AgelfiltrationchromatographyBHydroxylapatitechromatographyCHydrophobicinteractionchromatographyDAffinitychromatographyEIon-ExchangeChromatographyAgelfiltrationchromatography(1)mechanismAmolecularsizeBmobilesolventphaseCstationaryphase(2)Rigidityofbeadsinlargescaleoperation(3)traditionalgelfiltrationchromatographymediaASephadexBSuperoseCSephacrylDTrisacrylEBiogelPFCellulofineGFractogel(4)lowproductvolumesuitableforgelfiltration(5)activitylossAfinalfractionationBpreviousbioseparationstepsExmaple3.1carboxylesterasepurification

(1)Material：Bacillusstearothermophilus噬熱脂桿菌(2)SteputilizedAcentrifugationBDEAE-Sephacelion-exchangechromatographyCgelfiltrationchromatography(3)ResultanalysisAsinglelow-mobilityband(PAGE)BTwobandsofhighermobility(afterprecipitationandanionexchange）

Cjustsinglebandofhighmobility(furthergelfiltration)DgelfiltrationandSDSindicateesteraseismonomericproteinwithMW40000Exmaple3.1carboxylesterasepurification

(4)ReasonAactivemonomerassociatingmultipleformsBmultipleformhavedifferentchromatographyandelectrophoreticpropertiesCreactionequilibriumaffectedbypH,saltconcentration,enzymedilutionDmonomersormultimericenzymeformsaffectactivityandstabilityDifferencebetweencrudeandpurifiedesteraseforms(1)Thiol-containingcompoundsincrudematerialleadtocysteine-containingproteindenaturation(2)Proteolysisactivityincrudematerial(3)Coprecipitationwithlessstablecompoundsincrudematerial(4)ConformationalchangesinprocessingstepsResultsanddiscussionabouttable3.1(1)48%recoveryishighyield(notsohighpurificationfactor)(2)Statechangeofenzymeaggregationleadtoactivityloss(3)AnalyzecausepermitminimizeactivitylossPurificationofantigenizedimmunoglobulinswithmonomethoxypolyethyleneglycol(1)polyethyleneglycol(PEG)usageAnontoxicBnonimmunogenicCinternal（內(nèi)用）useinhumansDPEGylatedproteinspreservebiologicalactivity(2)variousdegreeofderivatizationoccurAproteinmicroheterogeneityBdistributionofnumberandportionofPEGCPEGpolydispersity(3)ChemicalreactionmPEGattachedtoAIgsPurificationofantigenizedimmunoglobulinswithmonomethoxypolyethyleneglycol(4)PurificationresultofAIg-mPEGusingsizeexclusionchromatographyAammoniumhydrogencarbonateasbuffersystemBUltrogelAcA-44gelcolumnCtwoelutionpeaksincludingAIgs-mPEGsandunconjugatedAIgs,andmPEG(5)FurtherfractionatingfirstpeakusingQ300anion-exchangecolumnAfirstpeakwithhighPEGylatedIg-Hemaglutinin(HA)BsecondpeakcontainingmildlyPEGylatedIg-hemaglutininCthirdpeakunconjugatedcontrolIg-HA(6)6-8%degreeofderivatizationandlonghalflife

Example3.2Tissueplasminogen

activatorpurification

(1)material：animalcellandbacterial(2)tPA2200USD/dose20timesthanstreptokinase(3)streptokinaseuseonlyonce,tPAmorethanonce(4)tPAmodelproductAflagshipproductforbiotechnolotyBfirstproductformarketusinggeneticalllyengineeredmammaliancellsinsteadofrecombinantbacteria(5)marketrequirementwith11000gtPA/YeartPApurificationprotocols(1)Affinitychromatography(2)Ion-exchangechromatography(3)Aminoacid–Sepharose(4)Gelchromatography(5)Ultrafiltration(6)Centrifugation(6)Microfiltration(7)Solubilization(8)Cleavage(9)refoldingt-PAfromCHOandE.coli(1)5stepspurificationforCHOand16stepsE.coli.(2)Affinitychromatographyandgelchromatographyusedintheendofpurificationprocesstoremoveimpuritiesandsalts(3)t-PApuritygreaterthan99.5%(4)Clearanceofendotoxinwith99.999%(5)Dropinproductyield(refolding20%,ultrafiltration56%Xylanasepurification(1)material:thermophilicascomycete(子囊菌)(2)xylans(majorhemicelluloseinangiosperms)(3)threeseparationstepsAultrafiltrationstepBanionexchangestep(Q-sepharosecolumn)Cgelfiltration(superose12column)(4)xylanaseinteractwithgelfiltrationtoresultinconformationalchange(hightyrosinecontent)PurificationoftwoisoenzymesofxylanasefromstreptomycesPurificationsequence(1)ammoniumsulfate(2)precipitation(3)centrifugation(4)desalting(SephadexG25)(5)DEAE-SepharoseFFcolumn(6)CM-Sepharosecolumn(7)concentrationbyultrafiltration(8)gelfiltrationonSephadexG75column(9)freeze-dryPurificationofhighlythermostableglucoseisomerase(1)material:thermohilicbacteriumThermotogamaritima（2）55%fructoseproducedat95-100C（3）purificationstepAcentrifugationBQ-SepharosecolumnCPhenyl-650MhydrophobicresincolumnDQ-SepharoseHPanionexchangeEHiloadsuperdex200gelfiltrationchromatography(4)SDSASinglebandBWithmolecularweight45000CHomotetramericstructureDMostcommonoligomericstateofglycoseisomeraseExample3.3purificationoftwoendo-β-glucanasefromaerobicfungus(1)material:aerobicfunguspenicilliumcapslatum(2)glycans(聚糖）problemsinbeerindustryAprecipitateBhazes（混濁）

CgelinstoredbeerDgumminess(3)PurificationstepAfreeze-dryofcrudeextractBgelfiltrationonaSephacrylS-300columnCionexchangeonaDE-52celluloseDultrafiltrationEelectrophoresis(4)endo-β-glucanaseAandendo-β-glucanaseB(5)gelfiltrationusedearlier(6)crudeextractismorestableandthepurifiedenzyme.Purificationprotocolforβ-glucosidasefromclostridiumthermocellum(1)material:clostridium(梭菌)therocellum(2)degradationofcrystallinecelluloseproducecellobiosebycellulasecomplex(3)β-glucosidasemetabolizecellobiose(4)purificationstepsAsonicationBcentrifugationCDEAE-SepharoseCL-6BchromatographyDionexchangechromatographyEhighperformanceNonoQHRcolumnFultrafiltrationGchromatofocusingonMonoPHRcolumnHgelfiltration(5)homogeneousenzymeobtainedPectinmethylesterasepurification(1)material:bacillussubtilis(2)pectinismajorstructuralcomponentinplantcellwalls(3)pectinasedegradepectin(4)PMEusageAclarificationofciderBdeesterificationofpectintomethanolandpectate(5)purificationstepsAcentrifugationBultrafiltrationCtwogelfiltration(6)homogeneousPMEobtained(7)proteinlossbyleakageinUFmembrane(PME36000Da,molecularcutoff10000Da)(8)lowactivityinabsenceofsalts(nonspecificbindingoraggregationHydroxyapatitechromatography羥基磷轔石(,Ca10(PO4)6(OH)2)（1）純化蛋白質(zhì)的無機介質(zhì)。（2）廉價，易于大規(guī)模應用，（3）使用后易于清潔吸附機理與規(guī)律：偶極離子作用而不是離子交換作用(1)每一個正電荷區(qū)周圍都有負電荷區(qū)，反之亦然.

（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。工藝條件吸附：(1)pH在6—9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）鹽可以采用恒定或梯度形式應用。BHydroxylapatitechromatographyExample3.5purificationofclostridiumthermoceilumβ-glucosidaseBβ-glucosidaseBusageCatalyzehydrolysisofβ-glucosidelinkagesbetweenglucoseandalkyl,aryl,orsaccharidegroupBioseparationprecedures

AammoniumsulfateprecipitationBQ-sepharoseFFcolumnCbutyl-sepharoseDhydroxylapatitePurificationresultAtable3.9Bstabilityincreasedbyadditionofdivalentcationat45-60CChromatographymediaSilicagelAregularshape(sphere)Buniformsize(3-10μm)CuncoatsurfacedissolvedbyalkalinesolutionDalkylgroupstendtobecleavedinacidicsolutionOthermaterialAtitaniaBzirconia硅膠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)（1）化學組成SiO2.XH20，（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。（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面。硅膠的分類（常以孔徑大小來分），(1)特細孔硅膠(0.8nm以下)。

(2)細孔硅膠（1.5—2.0nm)(3)中孔硅膠(4.0一5.0nm)(4)粗孔硅膠(10.nm以上)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SorbitolandsorbitoldehydrogenaseSorbitolapplication:sweetenerandfoodadditiveSorbitoldehydrogenaseapplication(oxidizessorbitoltoD-fructose)Sorbitoldehydrogenasepurification

A(NH4)2SO4precipitationBQ-SepharoseChromatographyCprocion-blueaffinitychromatographyDhydroxylapatitechromatographyEultrafiltrationPurificatonresultAtable3.10BsingleproteinbandbySDSCpurifiedenzymequitestableandstabilityincreasebyadditionofsucrosePurificationofD-xylulokinaseMaterial:yeastpichia(畢赤酵母屬)stipitisD-xylose:ThesecondlargestamountofsugarisnaturallyavailableD-xylullokinase（木酮糖激酶）

convertD-xylosetoethanolPurificationprotocol:TwostepshydroxylapatitechromatographyUltrafiltrationPurificationresultAtable3.11BhomogeneitysampleCmajoractivitylossoccurinthefirstadsorptioncolumnDpartiallypurifiedenzymeunstableat4C,butstableat-20CEallkinasereactionrequiredivalentmetalion(Mg2++)NewwordsandvocabulariesChitanase甲殼素酶Confectioning調(diào)制部分,調(diào)糖膏）Entail使必須，使承擔Rigidity剛性Carboxylesterase羧酸酯酶Esterase酯酶Mobility遷移率Status狀況Multimeric多聚體的NewwordsandvocabulariesThiol硫醇

cysteine半胱氨酸Underscore劃線，強調(diào)MonomethoxypolyethyleneglycolMicroheterogeneity微觀異種性Polydispersity多分散性Hemaglutinin凝集素Conjugation結(jié)合物Halflife半衰期Tissueplasminogenactivator組織纖維蛋白酶原激活劑Streptokinase鏈激酶NewwordsandvocabulariesBovinegrowthhormone牛生長激素Chinesehamsterovary(CHO)中國鼠卵細胞Cleavage裂解，分開Xylanase木聚糖酶Ascomycete子囊菌屬Hemicellulose半纖維素Angiosperm被子植物Lignin木質(zhì)素Tyrosine酪氨酸Xylanhydrolase木聚糖水解酶Actinomycetes放線菌NewwordsandvocabulariesSweetener甜味劑Fructose果糖Highfructosecornsyrup高果糖漿Hemotetrameric均一的四聚體Haze混濁Gumminess樹膠，粘性Pectin膠質(zhì)Pectinase膠質(zhì)酶Texture結(jié)構(gòu)，質(zhì)地Cider蘋果酒Flagellin鞭毛蛋白Hydroxylapatite羥基磷灰石Questions1Whatismechanismofgelfiltration?2Whichdifferencesbetweencrudeproteinsandpurifiedproteins3WhysomeenzymesweremodifiedwithmPEG?4Whystreptokinaseusedonlyonce,whiletPAusedmorethanonce?5Whatismechanismofhydroxylapatite?6Keywords:polydispersity,hemaglutinin,ChineseHamsterovary(CHO),highfructosecornsyrup,hydroxylapatite.

Chapter3

2stHydrophobicchromatographyExample3.7purificationofferuloy/p-coumaroylesteraseMaterial:funguspenicilliumpinophiliumXylans:majorconstituentsofwoodsandagriculturalresiduesP-coumaricesterase:esterifiestheL-arabinoseresiduesinthexylanbackbone(importantmodificationPurificationprocedures:AAmiconultrafiltrationBDEAE-SepharoseCL-6Banion-exchangechromatographyChydrophobicinteractioncolumnPurificationresultAtable3.12BSinglebandbySDSand57000DatonMWCstabilefrom37-55C疏水層析

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)介質(zhì)配基密度:40—80μmol/ml膠,吸附容量：牛血清蛋白(BSA)大于40mg/ml疏水層析介質(zhì)制備過程示意圖工藝條件吸附：(1)鹽濃度：高鹽，1—2M(NH4)2SO4

或NaCl(2)pH:中性或偏酸性淋洗：0.5—2%表面活性劑洗脫:(1)低鹽，0.1—0.5MNaCl,再配合pH變化。(2)低濃度促溶劑,0.1—0.5%NaSCN,或20—40%乙二醇疏水層析實驗方案：NewwordsandvocabulariesHydroxylapatite羥基磷灰石Alkyl烷基Aryl芳基Saccharide糖類Butyl-Shpharose丁基-SepharoseSilicagel硅膠Uncoated未涂層Titania二氧化鈦Zirconia氧化鋯Sorbitol山梨醇D-glucitol葡萄糖醇Acyclicpolyol無環(huán)多醇Nicotinamideadeninedinucleotide(NAD)尼克酰胺腺嘌呤二核苷酸Xylitol木糖醇Sucrose蔗糖D-xylulokinase木酮糖激酶Pentose戊糖Phosphorylation磷酸化Feruloyl/p-coumaroylesterase（氯苯二酚氨醇香豆素脂酶）Arabinose樹膠醛糖，阿拉伯糖AffinitychromatographyExample3.8purificationofchitanaseMaterial:trichoderma（木霉）barzianumPurificationprotocolAammoniumsulfateprecipitationBQ-Sepharoseion-exchangechromatographyCSephadexG-100gelfiltrationDphenyl-SepharoseCL-4BhydrophobicinteractionchromatographyPurificationresulttable3.13ClustermodelofmultivalentaffinitySelectivityisdependentonligandandmatrixMultivalentinteractionAtwoormoreligandinaclusterbindingtwoormorecontactsBcooperativityduetoproximityandentropicCassociationconstantsforligandpairs100-foldgreaterthanconstantforsingleligand,whilemodelconstantshouldbesevenordersofmagnitudehigherDgeometrical-stericconstraint

親和層析親和層析(AffinityChromatography)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。生物體內(nèi)相互作用的分子對：(1)酶—底物或抑制劑(2)抗原—抗體。(3)激素—受體。(4)糖蛋白與凝集素,(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)

活化(activation):

基質(zhì)（matrix）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)。(1)大分子配基如蛋白質(zhì)等可與活化基質(zhì)直接偶聯(lián)。(2)小分子配基,

在基質(zhì)與配基之間插入若干碳原子手臂(spacer),然后再與配基偶聯(lián)。(3)環(huán)氧氯丙烷,1,4－丁二醇縮水甘油醚本身是活化劑亦具有手臂作用?；罨椒ǎ?）A溴化氰法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)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,特別適用于偶聯(lián)敏感性生物大分子。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）B環(huán)氧氯丙烷活化

（1）所形成的共價鍵穩(wěn)定,配基不易落脫（2）自動引入手臂,（3）有更小的非特異性吸附,（4）活化操作簡單易行,危險性相對較小缺點：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-exchangechromatographyExample3.10purificationofk-carrageenaseMaterial:pseudomonas（假單孢菌屬）carrageenovoraCarrageenans（角叉菜聚糖）:majorcomponentofcellwallstructureinredalgaeCellwalldegradingenzyme:usedforobtainingprotoplastsandunderstandingstructureofcarrageenansPurificationprotocolSmallscaleA(NH4)2SO4precipitationBSephadexcolumngelfiltrationCCM-sepharoseCL-6BcolumnLargescaleAultrafiltrationBS-SepharoseFFcationexchangePurificationresult

Atable3.14Bdoubleband(32000and34000Da)離子交換法概述：利用離子交換樹脂分離生物物質(zhì)的方法特點：（1)濃縮效應

(2)成本低，設備簡單，操作方便

(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)高分子化合物骨架活性離子強酸性陽離子交換樹脂-SO3H，-PO（OH）2-PHO（OH）RSO3H+NaClRSO3Na+HCl交換能力與pH無關(guān)弱酸性陽樹脂-COOH，-OH(酚羥基）

強堿性陰樹脂

弱堿性陰樹脂Example3.11Productionofbloodprotein

usingion-exchange

Material:humanplacentalbloodandhumanplasmaMatrix:silicaparticlesArigidityBincompressibilityClowpressuredropDlargeporediameterEcoatedbysuitablepolymerssuchaspolysaccharideFpossibleapplicationonanindustrialscaleResultsanddiscussion

AProductionscale20000liters/dayand140KgofproteinBSucroseandalbuminminimizedenaturationofbloodcoagulatingfactorCExtensivedialysisordiafiltrationleadtodenaturationofproteinswhenremovalofcompetitiveligandsDMildelutionconditionswithmetalchelatingagentsminimizemetalleakage離子交換提取蛋白質(zhì)傳統(tǒng)離子交換劑不適用于提取蛋白質(zhì)（1）交聯(lián)度大（大分子不能進入）（2）電荷密度高(結(jié)合太強）（3）骨架憎水性強（蛋白質(zhì)易變性)親水性離子交換劑（1）親水性大（2）孔徑大（3）體積隨pH及離子強度變化小親水性離子交換劑功能團離子交換劑的交換容量親水性離子交換容量不能達到無機離子交換容量（1）蛋白質(zhì)不能進入活性中心（2）一個蛋白質(zhì)與多個功能基發(fā)生作用吸附機理

（1）靜電吸力（2）憎水（3）氫鍵（4）被吸附蛋白表面進一步吸附蛋白質(zhì)的離子交換層析層析介質(zhì)：DEAE-纖維素，CM-纖維素吸附條件：

(1)緩沖液pH(2)蛋白濃度控制（5mg/ml)（3）離子強度

(4)道南效應(Donnaneffect)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,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；cpH變化大NewwordsandvocabulariesAcetamido乙酰氨基Endoglycosidase葡萄糖內(nèi)酯酶Phenyl-SepharoseCL4B苯基-P-nitrophenyl-β-N-actylglucosamine對硝基苯-N-乙酰葡萄糖胺Entropic熵Rabbitmuscledehydrogenase兔肌脫氫酶Clusters簇Lactatedehydrogenase乳酸脫氫酶CibacronbluecelluloseOrdersofmagnitude數(shù)量級NewwordsandvocabulariesRandom隨機K-carrageenasek-角叉菜酶

algae海藻Seaweed海草，海藻Protoplasts原生質(zhì)體CM-SepharoseCL-6BPhycocolloid藻膠Placental胎盤的Chloroform氯仿Hemoglobin血紅素Electrolytes電解質(zhì)IIICrystallizationCrystallizationofbacterialluciferase（螢光素酶）Material:marinebioluminescent（生物發(fā)光）

bacteriumCrystallizationsteps(1)microfiltration(2)sitting-dropvapordiffusionmethodwithcrystallizationplates(3)Biomek1000automatedlaboratorysystem(accuracy,reproducibility,andprecision)Example3.12purificationandcrystallizationoflipaseMaterial:geotrichum（地絲霉屬）candidum（念珠菌）Lipasehaveahighspecificityforfattyacidshavingatleastonecis-Δ9doublebond.Purificationsteps(1)Q-Sepharosecolumn(2)Phenyl-SepharosecolumnCrystallization

(1)hanging-dropmethodusingmultichamberplates(2)crystallizedinthepresenceofPEG(3)crystalssizedependsonmolecularweightofcrystallizationagent(4)fivedifferentcrystalsobtaineddependingontheamountofglycosylation(5)pureenoughtoobtainX-raydiffractiondataPurificationandcrystallizationoflipasePurificationProtocol(1)ion-exchangechromatography(2)S-Sepharosefastflow(3)Phenyl-SepharoseCL-4Bhydrophobicinteractionchromatography(4)hydroxylapatiteadsorptionchromatography(5)Ω-aminohexylagaroseaffinitychromatographyCrystallization

(1)bundlesofneedlesobtainedinammoniumsulfite(2)rhombic(斜方）crystalsobtainedinammoniumsulfate(3)purecrystalweresuitableforX-raydiffractionstudiesAntibioticscrystallizationExample3.14penicillincrystallizationBasicbioseparationprotocol(1)filtration(2)centrifugation(3)liquid-liquidextraction(4)crystallizationDiscussion(1)purificationofantibioticsundersterilecondition(2)rapidprocessing(3)sensitivetotemperature(4)extracaretopreventcontaminationordegradation(β-lactamase-producingorganisms)Example3.15purificationandcrystallizationofcephalosporinAntibioticscrystallizationBioseparationprotocol(1)solventextraction(2)ion-exchangeresin(3)salting-out(4)activatedcarboncolumn(5)precipitation(6)crystallizationApotassiumorsodiumsaltofcephalosporinBmisciblesolventCCoper,nickel,orleadsaltcrystallizationfromaqueousssolutionsIVothertechniquesExample3.16purificationofβ-galactosidaseMaterial:Aspergillus（曲霉）fonsecaeusβ-galactosidase:hydrolysisoflactoseinmilkandwheyindairyindustryProtocol(1)ultrafiltraction-diafiltration(2)isopropanolprecipitationPurificationresult(1)table3.18(2)isopropanolprecipitationashigh-resolutionfractionationstep(3)diafiltrationtoremovemajorlowmolecular-weightcontaminants(4)furtherpurificationAhydroxylapatiteBgelfiltrationCanionexchange(Fastphaseliquidchromatography,FPLC)(5)bothpartialpurificationandhighpurificationprovidedExample3.17purificationofβ-glucosidaseMaterial:fungusNeocallimastixfrontalisEB188(1)Protocol(2)ConcentrationbyMiliporefilter(3)Ethanolprecipitation(4)Hydroxylapatitechromatography(5)Gelfiltrationchromatography(6)Ion-exchangechromatography(7)nativePAGEResultsandDiscussTable3.18HydroxylapatitechromatographytwiceAfirsttreatmentobtainedfourpeaksBsecondtreatmentobtainedtwopeaksExample3.18separationofperoxidaseMaterial:soybeanhulls（殼）