出芽短梗霉诱变选育及丙二酸抗性筛选聚苹果酸高产菌-毕业论文.docx

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/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Screening of high poly malic acid mutant of Aureobasidium pullulans against malonic acid of Succinodehydrogenase InhibitorAbstract ：Objective Starting from the AH2, by mutagenesis and screening to obtain poly malic acid production by Aureobasidium pullulans strains. Methods Taking AH2 as the starting strain, through nystatin pretreatment, changed by Aureobasidium cell membrane permeability, after UV mutagenesis of malonic acid resistance screening, and then by potassium and bromine phenol first green screen, according to the selection of culture based on the morphological characteristics of the colony and the HC value, select strains for fermentation. At the end of fermentation to produce poly malic acid was quantitatively determined and screened to obtain high-yield strains of PMA.Results Finally, a poly malic acid high yield strain 5-5-4 was obtained, its shake flask fermentation production reached 20.80g/L and the yield was increased by 6% compared with the starting strain AH2, and the yield was stable.Conclusion Aureobasidium pullulans of malonic acid tolerance can produce higher yield of poly malic acid by fermentation.This method overcame the blindness of random screening, and improve the efficiency of screening.Key words：Poly malic acid; PMA strain; potassium bromide phenol green screening; UV mutagenesis; screening of malonic acid resistance; Aureobasidium pullulans第1章 前言1.1出芽短梗霉 出芽短梗霉（Aureobacidium pullulans）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，菌落边缘呈现辖射状，菌落的质