嗜煙堿節(jié)桿菌煙堿脫氫酶性質(zhì)及其在煙草加工中的應(yīng)用研究.pdf

1、中國農(nóng)業(yè)大學(xué)博士學(xué)位論文嗜煙堿節(jié)桿菌煙堿脫氫酶性質(zhì)及其在煙草加工中的應(yīng)用研究姓名：馬林申請學(xué)位級別：博士專業(yè)：食品科學(xué)指導(dǎo)教師：孫君社20050601ABSTRA(玎Nicotine，asthemainalkaloid，iSupIo95percentintobaccoSmokingforalongtimewillmakeoneaddictivetonicotineandiIlllalillgnicotineexcessivelywill

2、preventcentralneurotransmitterfromtransferring，beparalytictoheart，andevenbedeathful_Soexactnicotinecontentmustbelabeledontobaccopackagebylawinmanycountries，warningthat’cigarettesmokingisharmfultooneshealth”Interestinglym

3、icrobeanditsmetabolizedenzymecandegradenicotine，whichishelpfulforhumanhealthimprovetllequalityofsmokeandutilityoftobaccoleavesandbringalotofeconomicbenefits(1)Selectionandidentificationofamicroorganismeffectivetodegraden

4、icotine：SomestrainsproducingnicotinedehydrogenaseO奶田wereisolatedfromsoilbyselectionmediumwithnicotineasthesolecarbonsource，andahighnicotinedegradationstrain(Z3)wasselectedbyexperimentsinshakeflasksThemorphological，physio

5、logicalcharacteristicsandsequenceof16srRNAofthestrainshowedthatitwasArthrobacternicotinovorans73，andthe16srRNAofthestrainandthatofArthrobacternicoanovoranswere978％homogenous(2)OptimizationofNDHproductionbyAnicotinovorans

6、Z3inshakeflask：Nicotinewastheoptimalcarbonsource，yeastextractand(NH)2S04weletheoptimalnitrogensourceTheactivityofnicotinedehydrogenasewasuptothegreatestafterculturing48hinrotaryshaker(220r／min)at30℃initialpH705％inoculumc

7、ulture(3)PurificationandcharacterizationofNDH：NDHwaspurifiedfromtheculturefiltrateofAnicotmovorans7_3byammoniumsulfateprecipitation，ionexchangechromatographyandgelfiltrationSDSPAGEelectrophoresisofthepurifiedfractionmiti

8、gatedasimpleband，whichrevealedthatthisenzymewaspurifiedandthemolecularweightofthisenzymewas126KDaResultsshowedthatoptimalactivityofNDHatpH70，temperature40。CDynamicstudiesdemonstratedthatitisanenzymewithKmof7694104mol／Lan

9、dV∽99510一mol／(minmg)Mn2andCo“canactivatetheenzymealthoughCu“inhibitit(4)CloningandexpressionofnicotinedehydrogenasegeneofAnicotinovorans：EscherichiacoliengineeredstrainwithhighyieldofNDHisobtainedNicotinedehydrogenasegen

10、eofAnicotinovoranswasclonedandconstructedintoexpressvectorPET17B，recombinantPET17B／ndhwastransformedintoEcoliBL21fDE3)pLYSandwashighlyexpressedTheengineeredstraincouldproducesolubleandactiverecombinantNDH，andtheyieldofit

11、sNDHwasasmuch25timesasthatof73strainRecombinantNDHwaspurified，anditiscomposedofthreesubunit(A，BandC)ofMr87KDa，30KDaandl5KDa(5)ConditionofnicotinedegradationbyNDHandsafetyevaluationofdegradingproduction：Nicotinewasdegrade

12、dbyNDHwithNADmethyleneblueor26dichlorophenolindophenolsupplementationinreactionsystemwhichwasanalycedbyHPLC，nicotinedecreasing4892％，1653％，1568％，respectivelyButnicotinewasnotconvenedinabsenceofelectronacceptorsAnimaltesta