26170.輔酶q,10高產(chǎn)菌株理性選育與發(fā)酵優(yōu)化

1、r_。。。?！?。。。。。。。。?！徽獜膶?shí)驗(yàn)室保藏菌中篩選到一株輔酶Qlo的產(chǎn)生茵XM023，產(chǎn)輔酶Q10達(dá)1880mg／L。首先通過(guò)菌落形態(tài)與細(xì)胞鏡檢觀察，以及液體培養(yǎng)與生理生化特征分析，初步判斷該菌為革蘭氏陰性菌，細(xì)胞富含色素，為典型的光合細(xì)菌。進(jìn)一步采用16SrDNA分子鑒定技術(shù)，測(cè)定XM02316SrDNA序列，并構(gòu)建了系統(tǒng)進(jìn)化樹(shù)，最終確定該菌為類(lèi)球紅細(xì)菌(Rhodobact

2、ersphaeroides)。以XM023為出發(fā)菌株，采用紫外線(xiàn)和LiCI兩輪誘變育種，篩選獲得耐對(duì)羥基苯甲酸、羅紅霉素的突變株XMII136，其輔酶Qlo產(chǎn)量為3179mg／L，較出發(fā)菌株提高6910％，突變株XMII136經(jīng)5次連續(xù)傳代培養(yǎng)，輔酶Q10產(chǎn)量保持在29mg／L33mg／L，結(jié)果表明XMII136是一株性狀較穩(wěn)定、可深入開(kāi)發(fā)研究的優(yōu)良菌株。對(duì)XMII136進(jìn)行輔酶Qlo發(fā)酵優(yōu)化，首先通過(guò)單因素篩選確定了培養(yǎng)基最佳碳源為葡

3、萄糖，氮源為玉米漿；最佳的發(fā)酵條件為：接種量10％，初始pH值68，裝液量50mL／250mL三角瓶；接著通過(guò)二水平PlackettBurman設(shè)計(jì)對(duì)培養(yǎng)基中的9種成分進(jìn)行篩選，獲得影響發(fā)酵的3個(gè)重要成分：葡萄糖、谷氨酸、NaCl；再利用響應(yīng)面分析法對(duì)該3組份進(jìn)行3水平的優(yōu)化，獲得它們的最佳組合：葡萄糖342mg／L、谷氨酸242mg／L、NaCI198mg／L。通過(guò)以上優(yōu)化過(guò)程，輔酶Qlo最終發(fā)酵水平達(dá)到12921mg／L，比優(yōu)化前提

4、高了306％。通過(guò)分析XMII136輔酶Qlo搖瓶發(fā)酵的代謝過(guò)程，結(jié)果表明輔酶Q10合成與菌體生長(zhǎng)為半耦聯(lián)過(guò)程?；贚ogistic方程和LuedekingPiret方程，構(gòu)建輔酶QIo發(fā)酵菌體生長(zhǎng)、底物消耗、產(chǎn)物生成的動(dòng)力學(xué)數(shù)學(xué)模型，對(duì)該動(dòng)力學(xué)模型進(jìn)行實(shí)驗(yàn)擬合，求解相關(guān)動(dòng)力學(xué)參數(shù)，最終獲得動(dòng)力學(xué)數(shù)學(xué)方程。該數(shù)學(xué)方程與實(shí)驗(yàn)數(shù)據(jù)能較好地吻合，較好地描述了XMII136輔酶Qlo搖瓶分批發(fā)酵過(guò)程。研究不同維生素對(duì)輔酶Qlo合成的影響，結(jié)果表

5、明：D胡蘿卜素效果最為明顯，比對(duì)照提高了323％。VBl、氯化膽堿、生物素分別比對(duì)照提高了112％、133％、108％：同時(shí)，實(shí)驗(yàn)表明不同添加物的最適添加時(shí)間也不同：生物素、Bl在發(fā)酵初期添加較好；而D胡蘿卜素、氯化膽堿在發(fā)酵過(guò)程中添加效果較好。此外，向培養(yǎng)基中添加豆油發(fā)現(xiàn)，豆油對(duì)輔酶QIo合成有促進(jìn)作用，其產(chǎn)量與對(duì)照相比提高了76％。對(duì)輔酶Qlo補(bǔ)料發(fā)酵進(jìn)行研究，首先，在搖瓶條件下考察了葡萄糖補(bǔ)加時(shí)間、補(bǔ)加量和補(bǔ)加方式對(duì)輔酶Qlo的影

6、響，結(jié)果表明發(fā)酵36h補(bǔ)加糖液比發(fā)酵起始補(bǔ)糖效果要好，補(bǔ)加總糖量為309／L，少量多次補(bǔ)加可獲得最大輔酶Qlo產(chǎn)量21878mg／L。在AbstractAbstractIIIIllIllIIIllllUlIIlY1806878ThestrainXM023producingCoQl0wasscreenedfromstrainspreservatedinthelaboratory，itsfermentationlevelofCoQt0WaS

7、upto1880mg／LFirstthroughtheobservationofcolonymorphology，cellmicroscopy，liquidcultureandbiochemicalcharacteristics，itwasfoundthatthisstrainWaSgram—negativebacteriawithrichpigmentincell，whichWaSpreliminarilyjudgedtobeonet

8、ypicalofphotosyntheticbacteriaThe16SrDNAofstrainXM023wassequencedandanalyzedforsimilaritieswithrelatedbacterialspeciesResultsshowedthatstrainXM023WaSamemberoftheRhodobactersphaeroidesXM023beingastartingstrainwasmutageniz

9、edtwicewithUVandLiClByscreeneingthemutantsresistingPHBandroxithromycinid，amutantXMII136WaSObtained，theCoQl0productionreachedto3179mg／L，whichwas69。10％higherthantheoriginalstrainAfterfivetimesofcontinuousculture，theyieldof

10、themutantWaSremainedat29mg／L一33mg／LResultsshowedthattheobtainedmutantXMII136wasstablestrainthatwasworthwhiletobestudiedfurtherThefermentationoptimizationofXMII136wasstudiedThroughsinglefactortestofmutantXMII136，theresult

11、showedthatthebestcarbonWaSglucose，theoptimumnitrogmWaStomsteeppowder；thebestfermentationconditionwasobtainedbasedonthesinglefactortest，itshowedtheoptimalamountofinoculationwas10％，theoptimuminitialpHwas68andtheoptimalliqu

12、idvolumewere50mLculturein250mLshakeflasksAP1ackett—Bunnandesignwasusedtoselect3importantfactorsfrom9impactfactorswhichaffectedCoQl0fermentationinculturemediumTheywereglucose，NaClandglutamicacidTheoptimumcomponentsobtaine

13、dbytheresponsesurfaceanalysismethod(RSM)wereglucose3429／L，NaCl2429／L，glutamicacid1989／LAftertheseoptimization，thefermentationlevelofXMII136reached12921mg／L，whichwas306％higherthanthatofnonoptimizedfermenatationBasedonthes