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1、出芽短梗霉的誘變選育及丙二酸抗性篩選聚蘋果酸高產(chǎn)菌摘要：目的：以出芽短梗霉（A.pullulans）AH2為出發(fā)菌株，經(jīng)紫外誘變和丙二酸抗性篩選獲得聚蘋果酸高產(chǎn)菌株。方法：以AH2為起始菌種，經(jīng)制霉菌素預(yù)處理，改變了出芽短梗霉細(xì)胞膜的通透性，再經(jīng)紫外線誘變后進(jìn)行丙二酸抗性篩選，然后經(jīng)溴鉀酚綠初篩，根據(jù)篩選培養(yǎng)基上的菌落形態(tài)特征以及HC值的大小，選出用于發(fā)酵的菌株，最后對發(fā)酵產(chǎn)生的聚蘋果酸進(jìn)行定量測定，篩選獲得PMA高產(chǎn)菌株。結(jié)果：最終獲得一株出芽短梗霉高產(chǎn)菌株丙5-5-4，其搖瓶發(fā)酵產(chǎn)量達(dá)到了20.80g/
2、L，比出發(fā)菌株提高了6%,且高產(chǎn)特性穩(wěn)定。結(jié)論：對丙二酸耐受的出芽短梗霉菌株可以高產(chǎn)聚蘋果酸，此種選育方法克服了隨機(jī)篩選的盲目性，提高了菌種篩選的效率。關(guān)鍵詞：聚蘋果酸；PMA高產(chǎn)菌株；溴鉀酚綠初篩；紫外誘變；丙二酸抗性篩選；出芽短梗霉ScreeningofhighpolymalicacidmutantofAureobasidiumpullulansagainstmalonicacidofSuccinodehydrogenaseInhibitorAbstract：ObjectiveStartingfromt
3、heAH2,bymutagenesisandscreeningtoobtainpolymalicacidproductionbyAureobasidiumpullulansstrains.MethodsTakingAH2asthestartingstrain,throughnystatinpretreatment,changedbyAureobasidiumcellmembranepermeability,afterUVmutagenesisofmalonicacidresistancescreening,
4、andthenbypotassiumandbrominephenolfirstgreenscreen,accordingtotheselectionofculturebasedonthemorphologicalcharacteristicsofthecolonyandtheHCvalue,selectstrainsforfermentation.Attheendoffermentationtoproducepolymalicacidwasquantitativelydeterminedandscreene
5、dtoobtainhigh-yieldstrainsofPMA.ResultsFinally,apolymalicacidhighyieldstrain5-5-4wasobtained,itsshakeflaskfermentationproductionreached20.80g/Landtheyieldwasincreasedby6%comparedwiththestartingstrainAH2,andtheyieldwasstable.ConclusionAureobasidiumpullulans
6、ofmalonicacidtolerancecanproducehigheryieldofpolymalicacidbyfermentation.Thismethodovercametheblindnessofrandomscreening,andimprovetheefficiencyofscreening.19Keywords：Polymalicacid;PMAstrain;potassiumbromidephenolgreenscreening;UVmutagenesis;screeningofmal
7、onicacidresistance;Aureobasidiumpullulans第1章前言1.1出芽短梗霉出芽短梗霉（Aureobacidiumpullulans）又稱為“出芽苗霉”、“黑酵母”及“短梗霉”等，是一種半知菌類短梗霉，是類酵母真菌，它歸屬于半知菌門、叢梗孢目、短梗霉菌屬具有酵母樣和真菌菌絲體兩種形態(tài)，這兩種菌體形態(tài)受培養(yǎng)基成分、培養(yǎng)條件等因素的影響，是可以相互轉(zhuǎn)變的。它在遺傳上具有不穩(wěn)定性，可以形成很多種變種，菌落最初有粘獨狀，呈臟白色，很快轉(zhuǎn)變?yōu)辄S色，之后變?yōu)樯罹G色，最后為黑色，菌落邊緣呈
8、現(xiàn)轄射狀，菌落的質(zhì)地開始較為粘稠，最后變得堅硬[[]LinYetal.Aureobasidiumpullulansbatchcultivationsbasedonafactorialdesignforimprovingtheproductionandmolecularweightofexopolysaccharides[J],ProcessBiochemistry,2007:820-827.]。出芽短梗霉能產(chǎn)生很
