超滤法纯化C群脑膜炎球菌荚膜多糖降解物的工艺优化周晖国

20188318ChinJBiologicalsAugust2018Vol.31No.81。2。、3730046C。300000C300kDa、1～6、0．15mol／LNaCl、0．5mol／LNaCl、50mmol／LPBSH2O、0．25、0．51mg／mL。C。C0．15mol／LNaCl51．0mg／mL。C80％。C。CR378．1＋5R392-33A1004-5503201808-0896-06OptimizationofpurificationprocedureforcapsularpolysaccharidehydrolyzateofNeisseriameningitidisgroupCbyultrafiltrationZHOUHui-guoZHANGHai-hongYUANFeiLUOShu-quanLIA-niYINGLian-fangLanzhouInstituteofBiologicalProductsCo．Ltd．Lanzhou730046Ga[e-mail]timizethepurificationprocessforcapsularpolysaccharidehydrolyzateofNeisseriameningitidisgroupCbyultrafiltration．MethodsThecapsularpolysaccharidehydrolyzateofN．meningitidisgroupCwaspurifiedbyultrafiltrationusingtheultrafiltrationmembranewithrelativemolecularmasscutoffof300000．Theultrafiltrationmembranematerial300kDapolyethersulfonemembraneandregeneratedcellulosecompositemembranetimesofdialysis1～6ultrafiltrationbuffer0．15mol／Lsodiumchloride0．5mol／Lsodiumchloride50mmol／LPBSandwaterinitialliquidconcentrationofpolysaccharide0．250．5and1mg／mLandultrafiltrationmethodconcentratedultrafiltrationandconstantvolumeultrafiltrationwereoptimizedusingmembranefluxandpolysacchariderecoveryrateasevaluationindexes．Thehydrolyzatepurifiedundertheoptimalconditionwastested．ResultsTheoptimalultrafiltrationmembraneultrafiltrationbuffertimeofdialysisinitialliquidconcentrationofpolysaccharideandultrafiltrationmethodforhydrolyzateofcapsularpolysaccharidesofN．meningitidisgroupCwereregeneratedcellulosecompositemembrane0．15mol／Lsodiumchloride5times1．0mg／mLandconcentratedultrafiltrationrespectively．Underthisconditionthepolysaccharidecomponentswithhighrelativemolecularmasseswereeffectivelyremovedwhilethepolysacchariderecoveryratewasmorethan80％andthepolysaccharidehydrolyzateendotoxincontentdecreasedtoacertainextent．ConclusionThepurificationprocedureforcapsularpolysaccharidehydrolyzateofN．meningitidisgroupCwassuccessfullyoptimizedwhichsignificantlyimprovedtheworkingefficiency．KeywordsUltrafiltrationNeisseriameningitidisgroupCCapsularpolysaccharideHydrolyzateProcedureopti-mization··[e-mail]896··DOI:10.13200/j.cnki.cjb.00226620188318ChinJBiologicalsAugust2018Vol.31No.8。。g／mol。1～2、34-5。。、、、。C、、、。11．1CHP-201604001。1．2NaAc、H2O2、NaCl、Na2HPO4、NaH2PO4LabscaleMilliporeAKTApurifier100GE。1．3RYALL6。50mmol／LpH6．0C10mg／mL0．5％H2O237℃2h10℃。1．450mgC20Psi20℃LabscaleJR。JL／m2·h＝V／A×TVLAm2Th。R％＝C1×V1／C0×V0C1V1mLmg／mLC0V0mLmg／mL。1．51．5．1300kDaC0．15mol／LNaCl200mL0．25mg／mL20mL0．15mol／LNaCl200mL4。、、。1．5．2300kDaC0．15mol／LNaCl200mL0．25mg／mL1～6。1．5．30．15mol／LNaCl、0．5mol／LNaCl、50mmol／LPBS0．15mol／LNaClpH7．2H2O200mL0．25mg／mL300kDa5。1．5．40．15mol／LNaCl0．25、0．51mg／mL300kDa5。1．5．5300kDaC0．15mol／LNaCl200mL1．0mg／mL5800mL0．15mol／LNaCl。1．6400mgC10kDa。、、1H1HNMR。1．6．1《》2015897··20188318ChinJBiologicalsAugust2018Vol.31No.87。1．6．2《》2015SepharoseCL-4B7。1．6．3《》20157。1．6．4NMR8。22．12．1．1300kDa300kDa80％。11。。1Tab1．EffectofmembranematerialonultrafiltrationpurificationL／m2·h％32389．2928581．982．1．2652。2Tab2．EffectofultrafiltrationtimesonmembranefluxandtherecoveryrateofpolysaccharidesL／m2·h％129046．63229066．16329675．10430480．12530883．17632083．952．1．30．15mol／LNaCl、0．5mol／LNaCl50mmol／LPBS0．15mol／LNaClpH7．284％H2O351L／m2·h23％。3。0．15mol／LNaCl。ABC。1CFig1．Effectofmembranematerialonultrafiltrationsepa-rationofcapsularpolysaccharidehydrolyzateofN．meningitidisgroupC3Tab3．EffectofvariousultrafiltrationbuffersonmembranefluxandrecoveryrateofpolysaccharidesL／m2·h％0．15mol／LNaCl29884．250．5mol／LNaCl29483．9850mmol／LPBS28484．28H2O35123．25－10．00．010．020．030．01.50.01.00.5150501000CmLmAUmAU－10．00．010．020．030．01.50.01.00.5150501000BmLmAUmAU1.50.01.00.5－0.5－1.0150501000－10．00．010．020．030．040．0AmLmAUmAU898··20188318ChinJBiologicalsAugust2018Vol.31No.82．1．40．25mg／mL0．51mg／mL3。4。、1mg／mL。4Tab4．Effectofinitialliquidconcentrationofpolysaccharideonmembranefluxandrecoveryrateofpolysaccharidesmg／mLL／m2·h％0．2529883．170．520888．66118481．582．1．567．8％262L／m2·h5。。5Tab5．EffectofultrafiltrationmethodonmembranefluxandrecoveryrateL／m2·h％30585．326267．82．21mg／mL10000～30000094．65％88．16％＞30000080．24％2．230．095EU／μg＞30000023．91EU／μg10000～30000080．79％。6。“”V0＞300000V0280nm2。1．30．9ppm3。6Tab6．Controltestsonpolysaccharidehydrolyzatebeforeandafterultrafiltrationpurificationmg％EU／μg％40088．162．23／＞3000005480．2423．9112．2910000～30194．650．09580．7930000093．08AB300000C300kDa。2CFig2．Molecularsizedistributionofpolysaccharidehydro-lyzatebeforeandafterultrafiltrationpurification－20．00．080．020．060．040．0100．00.05．04．03．02．01.0150501000mAUmL1.50.01.00.5－0.5－1.0150501000－10．00．010．020．030．040．0mAUmAUmLmAU－20．00．080．020．060．040．0150501000mAUmL1.50.01.00.5－0.5－1.02.02.5mAUABC899··20188318ChinJBiologicalsAugust2018Vol.31No.85.51.52.53.54.50.05．04．03．02．01.00.5ppmA125.51.52.53.54.50.05．04．03．02．01.00.5ppmB12AB300kDa。11．3ppm20．9ppm。3C1HNMRFig3．NMRspectraofpolysaccharidehydrolyzatebeforeandafterultrafiltrationpurification3。、、、、。C。3～5300kDa。HADIDI9300kDaUltracel8．4±0．1nm300kDaBiomax9．3±0．1nm1000kDaUltracel9．4±0．1nm。。。FOSCHIATTI10A。