出芽短梗霉的诱变选育及丙二酸抗性筛选聚苹果酸高产菌

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1、出芽短梗霉的诱变选育及丙二酸抗性筛选聚苹果酸高产菌摘要：目的：以出芽短梗霉（A.pullulans）AH2为出发菌株，经紫外诱变和丙二酸抗性筛选获得聚苹果酸高产菌株。方法：以AH2为起始菌种，经制霉菌素预处理，改变了出芽短梗霉细胞膜的通透性，再经紫外线诱变后进行丙二酸抗性筛选，然后经溴钾酚绿初筛，根据筛选培养基上的菌落形态特征以及HC值的大小，选出用于发酵的菌株，最后对发酵产生的聚苹果酸进行定量测定，筛选获得PMA高产菌株。结果：最终获得一株出芽短梗霉高产菌株丙5-5-4，其摇瓶发酵产量达到了20.80g/

2、L，比出发菌株提高了6%,且高产特性稳定。结论：对丙二酸耐受的出芽短梗霉菌株可以高产聚苹果酸，此种选育方法克服了随机筛选的盲目性，提高了菌种筛选的效率。关键词：聚苹果酸；PMA高产菌株；溴钾酚绿初筛；紫外诱变；丙二酸抗性筛选；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）又称为“出芽苗霉”、“黑酵母”及“短梗霉”等，是一种半知菌类短梗霉，是类酵母真菌，它归属于半知菌门、丛梗孢目、短梗霉菌属具有酵母样和真菌菌丝体两种形态，这两种菌体形态受培养基成分、培养条件等因素的影响，是可以相互转变的。它在遗传上具有不稳定性，可以形成很多种变种，菌落最初有粘獨状，呈脏白色，很快转变为黄色，之后变为深绿色，最后为黑色，菌落边缘呈

8、现辖射状，菌落的质地开始较为粘稠，最后变得坚硬[[]LinYetal.Aureobasidiumpullulansbatchcultivationsbasedonafactorialdesignforimprovingtheproductionandmolecularweightofexopolysaccharides[J],ProcessBiochemistry,2007:820-827.]。出芽短梗霉能产生很