歐文氏桿菌CXJZ95-198非纖維素降解特性及manA基因的克隆研究.pdf

1、中國農(nóng)業(yè)科學(xué)院博士學(xué)位論文歐文氏桿菌CXJZ95198非纖維素降解特性及manA基因的克隆研究姓名：張運(yùn)雄申請學(xué)位級別：博士專業(yè)：生物化學(xué)與分子生物學(xué)指導(dǎo)教師：劉正初20060601AbstractHerbaceousfiberextractedbiOl0窖icaUyisthedeve王opmentallrendw血ichcan咒ducetheheavYpoUu廿on，productcostari血gnomcl圯micalmetIlod

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3、fulstI丑inreportedfortllebio_extrac娃oⅡofherbaceousmaterialswitllin68hLTheuseofmono鞠cch缸id％andthewavnonccHulo∞de鯽dedi主Ithefemlentationproccsswercsfudiedinlegraledlyltsm口，一g曲ewassucce鼯fullyclo鵬d，蛆alyzed卸de)幣ressed1t’saddide

4、dtom啪oseM蛐nana∞isOneofthekeyfactorsinIllepmficient盟tfactionofherbaceousf如ersNoncelluloseinrawmatefialscanbedegradedbVthechainre虻tionof“noncelIulosenourishingmi啪organisfm—micmoIg卸ism∞cfetingenzynIHlIzymcdecOmposiIlgnoncel

5、lulose”afterinoctllationwithstrainsSugars，macromoleclllesandcastinthefe皿emationliquorincreaseaⅡdreachthep∞k68hrafterinoculation811％ofrawmatcrialcanbede哪dedintllefermentationcotlrse，90％0fo靴icsintheliquoraremacfom01ccuks柚d

6、c舔tThenOncellulo∞attachedtotllefibefscanbeeasⅡVremovedinthecomin衛(wèi)treahnentafteffermeⅡtationThefragm蛐tsmainlysized25kbafIer叩timizedpartialdigestion講tll踟“3舢couldbeu∞ddirectlyfortllegenomiclibraryconStIuction8positiVec10nes

7、wefespottedfmm15，o【)0tr卸sf0咖ants0nesmaⅡestncombinedpIasmidpT5of1844bpinscrtwas∞quencedItwasrevea王edtllatthekft7cIonesharbOred恤esamem口州g曲enw嬲dete衄inedthatthe13kbsequenceupstreamofIhe4，449，729lociofE翻陽幻vDmisthelocationfor州

8、d州geneTbisisthefirsttimet0clafify山efLlnctioⅡo“hegenomiclocjofEc口，D覘舊rnThe1137bpORFformn，“is0f10wsimnaritytooIherm咖如a畿genesfromdi血erent叫igins1t，sanewm口以geneTtlereisnotypicalpmlnotoffoundupstfeamoftheOREbutaposSibleSD∞qu蛐∞

9、existedontlle10catioⅡ_8negeneisre量isteredatGenB粕knumberedD036“40nIem肌4encodesfor387AAofmolecularweightof419KDAputativesignalpeptideexistsfrom1to27AAThesecondarystnlctureisof35％alphahdhand515％伯ndomcoil，andtwodomainsinclud

10、ingacatalyticonec卸befoundIt’sanextracellulareIlzyInecla船ified觴hydmlase26TheBlastshowsthatsomesimilaritiesexistbetweeⅡthecu玎如tmannanasesandthoseofotheroriginsThemannnanasesfromjkcf玎“sspafeofthellighestsimjl盯iliesamund5052

11、％衄dtheotherslessthan30％Expre船ingvectorswereconstnlctedandtheregIllationfactorswereoptimizedsi驢alpeptideisofh遠(yuǎn)himportancet0出e鯽灘tingofIheManAThemostsu“ableIPTGconcentfanonis1mM蛐dt11emostsuitabktemperaturcis37℃TwobandsofMwo

12、f42and47KDofhighdensitycanbefollndforVeclorswiIhs遠(yuǎn)nalpeptideand0ne伽getedbandc柚befolllldforVeclorswilhnosi驢alpeptideThe叩timalpHandtempefaturefortheexpfesscdManAis75蛐d50℃respectivelyKeywords：EcⅡrDfDvDr口，noncellulosedegradi