一种多级孔生物活性玻璃的制备及固载溶菌酶研究.pdf 10页

'中国科技论文在线http://www.paper.edu.cnThePreparationofHierarchicallyPorousBioactiveGlass#andImmobilizationofLysozyme*WangHongsu,LuoRuiping,ZhouXinrui5(CollegeofFoodScienceandEngineering,JilinUniversity,Changchun130062,China)Abstract:Hierarchicallyporousbioactiveglass(notedasYMBG)waspreparedbypinepollenforlysozymeimmobilizationviaintermolecularinteractions.ThemorphologyandporediameterdistributionofYMBGwerecharacterizedbyscanningelectronmicroscopy(SEM),transmittedelectronmicroscopy(TEM)andnitrogenadsorption-desorption.FTIR,UV,fluorescence,andcircular10dichroism(CD)studieswereshowedthatlysozymemaintaineditsoriginalsecondarystructureintheprocessoflysozymeimmobilization.Andtheoptimalexperimentalconditionswereexplored.Thismaterialpossessedahighlysozymeimmobilizedamount(648±5.1mg/gsupport).Inaddition,itwasfoundthattheadsorptionprocesswassuitablefortheLangmuirisothermalmodel.Andtheactivityofimmobilizedlysozymestillkeptabout52%oftheoriginalenzyme;theuseofimmobilizedenzyme15wasdistinguished,andtheactivityremainedabout25%afterusing4timesrepeatedly.Keywords:Materialscience,Hierarchicallyporousbioactiveglass,Lysozyme,Characterization0IntroductionHere,wereportedanovelapproachtoimmobilizelysozyme.YMBGwerepreparedbypine[1-3]20pollen,andthenuseitasacarriertoimmobilizelysozymebyintermolecularinteractions.Theoptimumconditionsofimmobilizelysozyme,suchasinitialconcentration,pH,adsorptiontemperatureandthenumberofoscillators(RPM),wereinvestigated.Theoptimumconditionswereobtainedandthenovelmaterialspossessahighlysozymeimmobilizedamount(648±5.1mg/gsupport).Theimmobilizedlysozymehasahighactivityandismaintainedatabout52%of25theoriginalenzyme,anditholdsahighutilizationrate,theactivityremainedabout25%afterusing4times.1Materialsandmethods1.1MaterialsPinepollenpowderwaspurchasedfromcommerciallyavailable;lysozymeandP12330triblockcopolymer(Poly(ethyleneglycol)-block-poly(propylene)-poly(ethyleneglycol))wereobtainedfromUnitedStatesSigma;triethylphosphate(TEP)waspurchasedfromShanghaiGuoyaoGroup;Calciumnitratetetrahydrate,anhydrousethanol,formaldehydesolution,concentratedsulfuricacid,concentratedhydrochloricacid,ethylorthosilicate(TEOS),abovechemicalreagentswerepurchasedfromBeijingChemicalFactoryreagentcompany.Ultra-purewaterwith3518.2MΩ·cmresistivitywasusedinallexperiments.1.2Materials1.2.1PreparationofMBGandYMBGTheMBGwerepreparedasfollows.10gofP123,18mlofTEOS,1.17gofcalciumnitratetetrahydrate,1.71mloftriethylphosphate,5mlofhydrochloricacid(0.5M)weredisperesedin[4]40ethanol(189ml),stirringfor24h.Themesoporousbioactiveglasswasobtained.AndtheYMBGwerepreparedasfollows.5gofpinepollenwasdispersedinethanol(50mL)Foundations:thePh.D.ProgramsFoundationofMinistryofEducationofChina(grant20130061120116).Briefauthorintroduction:HongsuWang(1978-),Famale,Associateprofessor,Immobilizationofenzyme.E-mail:wanghs@jlu.edu.cn-1- 中国科技论文在线http://www.paper.edu.cnunderultrasonictoprepareaqueousdispersion(0.1g/ml).Thewashedpollenwastransferredtoasolutionofethanolandformaldehyde(1:1).Andthenpollenwaswashedrepeatedlywithdeionizedwater.Finallycarbonizedwithconcentratedsulfuricacid.Theimmobilizedpollenwas45addedto50mlofconcentratedsulfuricacid(12M),stirredat80°Cfor4h.Whenthesolutionwaschangedfromyellowtoblack,filteredandwashedrepeatedlywithdeionizedwateruntilpH=7,thematerialwasdriedinadryingovenat80°Cfor12hours.ThenthedriedmaterialwasimpregnatedwiththeliquidMBG,repeating15times,dryinginanovenat30°C,andfinallyplacingthematerialinaresistancefurnaceandcalcinedat700℃for8h.Themulti-level[5]50bioactiveglassmaterialwasobtained.1.2.2AdsorptionanddesorptionoflysozymefromYMBGForthemeasurementstheadsorptionequilibriumofYMBG,10mgofYMBGwasaddedtoatransparentglassbattlecontaining4mloflysozymesolutionwithdifferentconcentrationsatvariouspHsvalues.Adsorptionexperimentswereconductedfor2hinashakingincubatorwith55variousshakingrateataconstanttemperature.Afterreachingadsorptionequilibrium,thesupernatantwasanalyzedbytheUV-2550spectrophotometer(SHIMADZU,Japan)at280nm,andtheadsorptioncapacityofYMBGforlysozymeadsorptionwascalculatedaccordingtothefollowingequation.V（C−C）0eq=m60where,qistheadsorptioncapacityofYMBGtolysozyme(mg/g),C0istheinitiallysozymeconcentrationoflysozymesolution(mg/ml),Ceisthelysozymeconcentrationatequilibrium(mg/ml),mistheamountofYMBG(g)andVisthevolumeofthelysozymesolution(ml).Theadsorbedanddriedcarriermaterialwassubjectedtoanelutiontest.ThemultiwellcarrierwasplacedinPBS(0.01M,pH=7.2-8.0,1MNaCl),shakenfor1hat37°Cinaconstant65temperatureoscillator,andfinallytheeluatewascollected.1.2.3CharacterizationsThemorphologyandsizeoftheYMBGwerecharacterizedbyscanningelectronmicroscopy(SEM)andtransmissionelectronmicroscope(TEM).Fouriertransforminfrared(FTIR)spectrawasobtainedbytheconventionalKBrpellettechniqueonaIRPrestige-21spectrometer70(SHIMADZU,Japan).TheN2sorptionisothermswasusedtocharacterizetheporediameterdistribution.ThesecondarystructureoflysozymeafterimmobilizationanddesorptionwasdeterminedbyUV-2550spectrophotometer(SHIMADZU,Japan),fluorescence(SHIMADZU,Japan)andCD-500（Biologic,France).1.2.4Determinationofenzymeactivity75Thestrainswerefreeze-driedpowder,andwereusedafterresuscitationandactivationintheexperiment.OD450wasadjustedto1.3bydilutingthebacterialsolutioninPBS(0.1M,pH=7.2-8.0).10mgoftheenzyme-carryingporouscarrierdissolvedin25mLoftheabove-mentionedbacterialsolution.Theamountofabsorbancedecreasedperminutewasmeasuredbymicroplatereader.LysozymeactivitycanbecalculatedusingEquation:UInitialabsorbance-Endabsorbance80Lysozymeactivity（）=mg0.001×t×Enzymequality-2- 中国科技论文在线http://www.paper.edu.cn2ResultsanddiscussionYMBG2.1PreparationandcharacterizationsofYMBGScanningelectronmicroscopy(SEM)andtransmissionelectronmicroscopy(TEM)wereused85toinvestigatethemorphologyandmicrostructureofYMBGobtained(Figure.1).ThelowmagnificationSEMimage(aandb)revealsthatthemorphologyoftheYMBG.Itdemonstratesthattheporousmaterialssuccessfullyreproducedthemorphologyofpinepollen.TheTEMimage(Fig.2)showsthatthemesoporousstructureoftheYMBG,atwo-dimensionalhexagonalcrystalstructure,aporesizeofabout6nm.Andinadditiontothemesopores,somemacroporesare90observedextrudingontheoutersurfaceofYMBG.Fig.1TheSEM(a,b)andTEM(c,d)ofYMBGThetypicalnitrogenadsorption/desorptionisothermsandthecorrespondingpore-size95distributionoftheYMBGareshowninFig.2AccordingtotheInterna-tionalUnionofPureandAppliedChemistry(IUPAC)nomen-clature,theresultingisothermcanbeclassifiedasatypeIVisothermwithtypeH3hysteresis,theinflectionpointappearsatarelativepressureofabout0.7,indicatingtheexistenceofmesoporousstructureandsomeslightlylargerpores.Thecorrespondingporesizedistributioncurvecalculatedfromthedesorptionbranchofnitrogen100isothermsbytheBJHmethodshowsthediameterisabout30-60nm.-3- 中国科技论文在线http://www.paper.edu.cn800.16A700.14B600.12/gSTP3500.10400.080.0630dV/dD0.04200.0210VolumeAdsorbedcm0.000-0.020.00.20.40.60.81.010100RelativePressure(P/P)Porediameter(nm)0Fig.2TheN2sorptionisotherms(A)andporesizedistribution(B)ofYMBGThebioactiveglasscarrier,lysozymeandimmobilizedenzymematerialwerecharacterizedby-1Fouriertransforminfraredspectroscopy(FTIR)intherangeof400-4000cm(Fig.3).Itcanbe-1-1105seenthattheabsorptionpeakat467cmand1077cmstillexistsafterimmobilizationofthebioactiveglasscarrier.Andthecharacteristicabsorptionpeaksoflysozymewerefoundat-1-1-11530cm,1658cmand3315cm,indicatingthatlysozymewassuccessfullyimmobilizedonthe-1-1multistagebioactiveglasscarrier.Moreover,theabsorptionpeakat1530cm,1658cmdidnotshiftuponimmobilization,suggestingthatthesecondarystructureofimmobilizedlysozymewas110wellmaintained.ABCT(%)10773315165815304674000350030002500200015001000500-1λ(cm)Fig.3TheFTIRspectraof(A)lysozyme,(B)YMBGand(C)lysozymeimmobilizedYMBG.2.2ImmobilizationoflysozymetoYMBGTodeterminetheeffectfactorsandsoughttheoptimalloadingconditionsforlysozyme115immobilizationontoYMBG,weexploretheeffectsofinitialconcentration,PH,temperature,ionicstrength,RPM,asshowninFigure.4(a,b,c,d,e),respectively.TherelationshipsbetweentheinitiallysozymeconcentrationandtheadsorptioncapacityofYMBGareillustratedinFig.4a.Itwasfoundthattheadsorptioncapacityincreasedlinearlyinthelowerrangeofinitiallysozymeconcentration,inferringthatallthelysozymemoleculeswere120adsorbedontotheYMBG.However,theadsorptioncapacitystartedtodeviatedownwardfromthestraitlinewhentheinitialconcentrationcontinuouslyincreased,indicatingthatthemaximumadsorptioncapacityofYMBGwasobtainedat4mg/mL.Similarly,wealsoexploredtheeffectofPHontheadsorptionoflysozyme.ThechangesinadsorptioncapacityasafunctionofpH（4.0-11.0）wereplottedinFig.4b.Itcanbeseenthatthe-4- 中国科技论文在线http://www.paper.edu.cn125pHvaluehasagreatinfluenceontheadsorptioncapacityoflysozyme,andtheadsorptioncapacityreachesthemaximumatpH9.0.Theeffectoftemperatureonadsorptionwasinvestigated.Itcanbeobservedtheadsorptionreachthemaximumatat37℃inFig.4c.Inordertoinvestigatetheeffectofionicstrengthonlysozymeadsorptionprocess,wechosetoaddNaCltoadjusttheionicstrength.TheexperimentalresultsareshowninFig.4danditisobviousthattheadsorption130capacityismorethan1mol/Lwhentheionicstrengthis0mol/L,whichcanbeattributedtotheionicstrengthincreasewillcausetheproteintosaltingoutthereactionandoccuraggregationprecipitation.Finally,wealsoexploretheeffectofRPMonthefixedload.itisclearthattheoptimalRPMconditionis180rpminFig.4e.Withthemaximumadsorptioncapacityof648±5.1mg/gundersuchconditions,whichthe135optimalinitialconcentrationis4mg/mL,theoptimaltemperatureis37℃,theoptimumpHis9.0,theionicstrengthis0mol/L,thenumberofrevolutionsoftheoscillatoris180rpm.This[2]valueisgreaterthanthosereportedintheliterature,probablyduetotheuniqueporousstructureofYMBG.0.20700ba0.186000.165000.140.124000.10300q(mg/g)C(mg/ml)0.082000.060.041000.02002468103456789101112Time(h)pH700800dc30°C0mol/LNaCl60037°C7001mol/LNaCl42°C600500500400400q(mg/g)300q(mg/g)30020020010010000020406080100120020406080100120140160Time(min)Time(min)140650e600550500450q(mg/g)400350300406080100120140160180200220rpmFig.4Theeffectofinitialconcentration(a)、pH(b)、reactiontemperature(c)、ionicstrength(d)、RPM(e)totheimmobilizingprocess.-5- 中国科技论文在线http://www.paper.edu.cn2.3Adsorptionisotherms145Theadsorptiondatawerefittedwithmathematicalisothermalmodelstoobtainmoreunderstandingoftheimmobilizationprocessoflysozyme.BothLangmuirandFreundlich2isothermswereusedtodescribetheadsorptionbehavior.Thecorrelationcoefficient(r)wasobtainedtocomparetheapplicabilityofthetwoisothermmodels.TheLangmuircurveassumesthattheadsorptionprocessismonolayeranditoften150applicabletoahomogeneousadsorptionsurfacewithalltheadsorptionsiteshavingequal[6]adsorbateaffinifty,andisrepresentedbythefollowingequation:111=+qqqbCmaxmaxeWhiletheFreundlichisothermmodelassumesheterogeneityofadsorptionsurfaces,155expressedbythefollowingequation:1lnq=lnC+lnKeeFnWhere,Ceisthesolutionconcentrationoflysozyme(mg/mL),qeistheamountofadsorptionattheconcentrationequilibriumstate(mg/g),qmaxisthemaximumadsorptioncapacityatequilibrium(mg/g);bistheadsorptionconstant.160Fig.5andFig.6shownstheLangmuircurvesandtheFroldlichcurvesoflysozymeadsorptionbyYMBGatthethreetemperaturesrespectively.ThecorrelationcoefficientsobtainedfromtheLangmuirmodeltheFreundlichmodel,andit2canbeseenthattherintheLangmuirmodelishigher,indicatingthattheLangmuirmodelisamoreapplicablemodelforlysozymeadsorptionontoYMBG.A0.0930℃0.082R=0.93710.070.060.050.041/qe(g/mg)0.030.020.0100123451/Ce(ml/mg)165B0.03537℃20.03R=0.99560.0250.020.0151/qe(g/mg)0.010.005000.511.522.51/Ce(ml/mg)-6- 中国科技论文在线http://www.paper.edu.cnC0.0442℃0.0352R=0.99860.030.0250.020.0151/qe(g/mg)0.010.00500123451/Ce(ml/mg)Fig.5TheLangmuirisothermoflysozymeadsorptionontoYMBGatdifferenttemperatures.A730℃6R2=0.944854lnqe3210-2-1.5-1-0.500.511.52lnCe170B737℃62R=0.983254lnqe3210-2-1.5-1-0.500.511.52lnCe-7- 中国科技论文在线http://www.paper.edu.cnC742℃26R=0.998154lnqe3210-2-1.5-1-0.500.511.52lnCeFig.6TheFreundlichisothermoflysozymeadsorptionontoYMBGatdifferenttemperatures.2.4Conformationalstudies175Thisstudyalsoexploredthechangesinthesecondarystructureofenzymemoleculeswhentheyinteractonthesurfaceorporesofthemultistageporesbioactiveglassmaterial.ThespectralanalysisofthesecondaryconformationoftheenzymemoleculeswascarriedoutbyUV,fluorimeterandCD.AsshowninFig.7A,itcanbeseenthatthepeakpositionoftheoriginalenzymesolutionandthedesorbedenzymesolutionare280nm,andthereisnoobviousdeviation.180Andthesamespectralcharacteristicsarealsoshowninthefluorescencespectra(Fig.7B).CDspectrometerisusuallyusedtostudywhetherthesecondaryconformationofproteinchanges.ItcanbeseenfromtheFig.7Cthatthethepeakpositionoforiginalenzymesolutionandthedesorbedenzymesolutionare208nm,indicatingthattheamountofα-helixstructuredidnotchangemuch.185Aseriesofspectralanalysisandcorrespondingdatashowedthattheconformationofimmobilizedlysozymedidnotchangedandthebiologicalactivityoftheoriginalenzymehadbeenretained.A0.7a0.6b0.50.4Abs0.30.20.10.0240260280300320340360380400420wavelength(nm)-8- 中国科技论文在线http://www.paper.edu.cnB400a350b300250200150100fluorescendintensity500-50300320340360380400wavelength(nm)10Ca5b0-5mdeg-10-15-20-25200210220230240250260wavelength(nm)190Fig.7UV(A),fluorescenceemission(B),andCDspectra(C)ofnative(a)anddesorbedLysozyme(b).2.5DeterminationofenzymeactivityInaddition,theactivityofthestandardenzymeandtheenzymeactivitywas18400±53U/mgand11400±28U/mg,respectively,whichindicatedthattheimmobilizedenzymeactivity195remained61.9±0.2%.Theactivitystillhad56.5±0.4%oftheinitialloadingenzymeactivityand35.1±0.3%oftheoriginalenzymeactivityafter4timesofrepeateduse,indicatingthattheimmobilizedenzymehadbetterreproducibilityandcouldbeusedrepeatedly.Therefore,theuseofnewmaterials,YMBGforadsorptionoflysozyme,hasgreatadvantages,suchasadsorptioncapacity,highstability,immobilizedenzymeactivity,goodre-use,andYMBGcanalsobeusedto200otherareasofenzymeadsorption,withhighapplicationvalue.3ConclusionTosummarize,wehavesuccessfullyfulfilledthesynthesisofYMBGthroughthenaturalpinepollenasahardtemplate.Duetothecomplexstructureofthenaturaltemplates,thenanomaterialshavemanykindsofporestructures,whichcanrealizetheeffectivecombinationofdifferentpore205structuresandfunctions.Itcanbeusedasaexcellentadsorbentandhasagooddevelopmentprospectinenzymeimmobilization.ThenewYMBGdemonstrateanexcellentadsorptionperformanceaboutlysozymeinwater,withthemaximumadsorptioncapacityof648±5.1mg/gundersuchconditions,whichtheoptimalinitialconcentrationoflysozymewas4mg/mL,theoptimaltemperaturewas37℃,theoptimumpHwas9.0,theionicstrengthof0mol/L,theRPM210was180rpm.AndtheisothermaladsorptioncurvemodelwasstudiedandfoundLangmuircurve-9- 中国科技论文在线http://www.paper.edu.cnmodelmoresuitable.Moreover，Aseriesofdatashowthatthedesorbedlysozymepreserveditsoriginalsecondarystructure；andtheimmobilizedlysozymeactivitywasmaintainedat61.9±0.2%andretained35.1±0.3%oftheoriginalenzymeactivityafterusing4times,indicatingthattheimmobilizedenzymehasbetterreproducibilityandcanbeusedrepeatedly.Therefore,ourfindings215validatethatYMBGcanbeusedasanovelmaterialforefficientlysozymeimmobilization,andcanalsobeusedtootherareas.AcknowledgementsTheauthorsacknowledgethefinancialsupportbythePh.D.ProgramsFoundationofMinistryofEducationofChina(grant20130061120116).220References[1]HenchLL,SplinterRJ,AllenWC,GreenleeTK.Bondingmechanismsatinterfaceofceramicprostheticmaterials[J].JBiomedMaterRes,1971(2):117-141.[2]YanPH,WangJQ,OuJF,LiZP,LeiZQ,YangSG.Synthesisandcharacterizationofthree-dimensional225orderedmesoporous-macroporousbioactiveglass[J].MaterialsLetters2010(64):2544-2547.[3]FathiMH,MohammadiAD.Preparationandcharacterizationofsol-gelbioactiveglasscoatingforimprovementofiocompatibilityofhumanbodyimplant.Mat.Sci.Eng.A.,2008,474:128-133.[4]WangHS,GaoXH,WangYN,TangJL,SunCC,DengXM,NiuXD.Bio-templatedsynthesisofmesoporousbioactiveglasswithahierarchicalporestructure[J].MaterialsLetters,2012(76):237-239.230[5]XiaY,ZhangWK,XiaoZ,etal.BiotemplatedfabricationofhierarchicallyporousNiO/Ccompositefromlotuspollengrainsforlithium-ionbatteries[J].J.Mater.Chem.,2012(22):9209-9215.[6]YangS,WangY,XuM,HeM,ZhangM,RanD,JiaX.Synthesisofmodifiedchitosan-basedmolecularlyimprintedpolymersforadsorptiveproteinseparation.Anal.Methods.2013,5:5471-5477.235一种多级孔生物活性玻璃的制备及固载溶菌酶研究240王虹苏，罗瑞平，周欣蕊（吉林大学食品科学与工程学院，长春，130062）摘要：利用松花粉制备多级孔生物活性玻璃（YMNG），通过分子间的相互作用固定溶菌酶。通过扫描电子显微镜（SEM）、透射电子显微镜（TEM）和氮吸附-脱吸等温线对YMBG的形态和孔径进行表征，通过FTIR，UV，荧光光谱和圆二色谱（CD）研究表明固载后的溶245菌酶仍保持原有的二级结构。该实验还探究了最佳的固载条件，在此条件下具有较高的固载量为648±5.1mg/g。此外，研究发现该吸附过程符合Langmuir等温模型。固定化后溶菌酶的活性仍保持原酶的52%，反复使用4次后仍能保持25%左右。关键词：材料科学；多级孔活性玻璃；溶菌酶；表征中图分类号：Q599250-10-'