洛伐他汀高产菌株土曲霉的诱变筛选方法及发酵条件研究

【作者】 贾志华；

【导师】 张小里；

【作者基本信息】 西北大学 ， 生物化工， 2003， 硕士

【摘要】 土曲霉（Aspergillus terreus）是洛伐他汀（lovastatin）的主要产生菌，但一般其亲本菌株产洛伐他汀能力较低，极大地制约了洛伐他汀的工业生产。本文有鉴于此，通过紫外诱变处理，并基于粗糙脉孢酶（Neurospora crassa）对洛伐他汀的生物敏感性，对诱变菌株进行了初筛；同时，采用摇瓶培养，结合TLC和HPLC的分析方法，复筛出一株高产菌株；对复筛过程两种分析方法分别进行了初步探讨，比较其优劣，确定TLC法经溴酚兰显色剂显色处理为较佳方式；从而，建立了一套简单、实用、快速、经济且有效的洛伐他汀生产菌诱变筛选方法。 通过摇瓶培养，对发酵培养基组成进行了初步优化，确定了最佳碳、氮源及最佳碳氮比。甘油为最佳碳源，酵母膏和黄豆粉为最佳有机氮源，NH₄H₂PO₄则为最佳无机氮源。在碳源初始浓度较低时，采用低的碳氮比，有利于发酵产物洛伐他汀生成；而碳源浓度较高时，提高碳氮比，可增加产物产量。在优选条件碳源浓度为50g/L、碳氮比为2.5的条件下，得到发酵终了时洛伐他汀产量为448mg/L浓度。 研究了摇瓶培养条件下装液量、接种量等对菌丝球形成及洛伐他汀产量的影响。实验确定，接种孢子浓度增大，生成的土曲霉菌丝球数量多、直径小、分布均匀，产洛伐他汀能力大；孢子浓度在2×10⁷以上，效果较佳；而装液量则不宜太大，500 mL摇瓶装液量取15～20％较为适宜，证明，通气对产物合成的重要性。 以2L通气搅拌发酵罐中分批培养结果为基础，对土曲霉产洛伐他汀过程菌体生长和产物生成动力学进行了研究，初步建立了土曲霉生产洛伐他汀的分批发酵动力学模型，并对模型参数进行了估计，发酵动力学模型较好地预示实验结果。更多还原

【Abstract】 As a main lovastatin-producing fungi, the wild strain of Aspergillus terreus produces lower lovastatin, however, limiting its industrial production greatly. In this paper, an efficient and easy biological screening method based on the sensitivity to lovastatin of Neurospora crassa was built to deal with the mutant strain rudely by UV mutation. A high-level strain was screened in the shaking flask culture incorporating with TLC and HPLC. A comparison between this two methods also was originally discussed while identifying dyeing using bromophenol blue as the better treatment of TLC, and offering a kind of simple, practical, fast and economical determination qualitatively and quantitatively.An primary optimization for medium composition in shake flask showed the best carbon source, nitrogen source and its best ratio. The results demonstrate glycerol as the best carbon source, yeast extracts and soybean meal as the best organic nitrogen source, NH4H2PO4 as the best inorganic nitrogen source. A lower ratio can promote lovastatin production in lower original concentration of carbon source while increasing its ratio in higher level can also result in it. A 448 mg/mL end yields of lovastatin was obtained along with the best C/N ratio in 2.5 and the original carbon source in 50g/L.The effects of culture volume and incubating quantity on mycelium formation and lovastatin production in shaking flask was researched. The experimental data suggested higher spore density would form larger quantity mycelium possessing lower diameters and well-proportioned profiles which could produce high-level lovastatin. A 2+10 7spore/mL suspensions supplied a satisfied result and a 15-20% culture volume in shaking flask of 500mL-scale can also result in a better effect wherein a lower culture volume was suitable. These all testified the importance of aeration to synthesis of end-product.The dynamics of mycelium growth and end-product formation in the lovastatin production by Aspergillus terreus strain was studied in the 2L fermentor based on its batch culture results. At the same time, the batch fermenting dynamics model of lovastatin was also built and the model parameters were subsequently estimated, which indicated to the experimental results better.更多还原