产琥珀酸大肠杆菌工程菌WS102的构建、发酵及琥珀酸分离的研究

发布时间：2018-05-31 13:15

本文选题：大肠杆菌 + 琥珀酸　；参考：《湖北工业大学》2017年硕士论文

【摘要】：琥珀酸是一种非常重要的C4平台化合物是合成很多重要产品的中间化合物,以琥珀酸为基础的化学衍生产品在工业、农业、食品、医疗等领域有着巨大的应用价值。发酵法产琥珀酸以可再生能源为原料通过微生物转化的方法生产琥珀酸作为一种绿色环保的方法较传统的以石油为原料生产琥珀酸有着巨大的优势。本文在实验室已构建的大肠杆菌工程菌WS100的基础上,通过λRed同源重组系统,敲除其丙酮醛合成酶基因(mgsA),再通过无氧管培养得到菌株WS102,进一步消除副产物乳酸的代谢。通过NBS培养基发酵罐验证,得到的发酵液琥珀酸含量64.9 g/L,琥珀酸的质量收率为75.1%,且发酵液中无副产物乳酸检出,而WS100琥珀酸收率72.3%。在此基础上进行WS102利用二氧化碳发酵琥珀酸的研究,研究发现全阶段以二氧化碳气体发酵琥珀酸并不能很好的产琥珀酸,前期的厌氧条件容易造成代谢失衡,产生大量副产物乙酸,在NBS培养基中发酵琥珀酸产量和收率分别为52.65 g/L和68.9%。因此,通过单因素的比较设计一条微好氧-厌氧的发酵工艺,通过在前期添加KHCO_3保证菌体良好生长,在稳定期之前以K_2CO_3提供固定“CO_2”所需C源,通过搅拌使前期保持微好氧的状态,在稳定期以后通二氧化碳气体进行琥珀酸发酵的工艺。通过在NBS培养基中分批发酵得到琥珀酸产量为58.3g/L,琥珀酸的质量收率为73.1%,生产强度为0.972 g/L·h。在此基础上进行分批补料发酵,琥珀酸产量为76.5 g/L,琥珀酸的生产强度和质量收率分别为1.063 g/L·h和76.5%。本文最后以钙盐法分离提取发酵液中的琥珀酸,工艺路线为首先将发酵液通过简单的加热过滤除去大颗粒物质,然后通过陶瓷膜过滤去除绝大部分菌体,通过钙盐沉淀,活性炭脱色以及离子交换的方法分离提取发酵液中的琥珀酸。并且对钙化工艺做了优化,优化钙化反应条件为在80℃条件下添加等体积的30%氯化钙溶液反应30 min。最终得到琥珀酸结晶99.6 g,琥珀酸收率为56.8%。
[Abstract]:Succinic acid is a very important C4 platform compound which is a intermediate compound for the synthesis of many important products. Succinic acid based chemical derivatives have great application value in the fields of industry, agriculture, food, medical and other fields. Succinic acid is produced by succinic acid to produce succinic acid using renewable energy as raw material through microbial transformation. As a green environmental protection method, it has a great advantage over the traditional production of succinic acid with oil as raw material. On the basis of the constructed Escherichia coli WS100 in the laboratory, this paper knocks out its acetone aldehyde synthase gene (mgsA) through the homologous recombination system of lambda Red, and then through the anaerobic tube culture to obtain the strain WS102 and further eliminate the pair. The metabolism of lactic acid was verified by NBS culture medium fermentation tank, the content of succinic acid was 64.9 g/L, the yield of succinic acid was 75.1%, and no by-product lactic acid was detected in the fermentation broth, while the WS100 succinic acid yield 72.3%. was based on WS102 utilization of carbon dioxide in the study of succinic acid, and the study found that the whole stage was two oxygen. Succinic acid fermented by carbon gas can not produce succinic acid well. The anaerobic conditions in the early stage can easily lead to metabolic imbalance and produce a large number of by-products acetic acid. The yield and yield of succinic acid fermented in the NBS medium are 52.65 g/L and 68.9%. respectively. The addition of KHCO_3 ensures good growth of the bacteria and provides the C source for the fixed "CO_2" before the stabilization period. The process of succinic acid fermentation by carbon dioxide gas is carried out after the stabilization period by stirring to keep the state of microaerobic at the early stage. The yield of succinic acid is 58.3g/L, succinic acid by the wholesale fermentation in the NBS medium. The yield was 73.1% and the production intensity was 0.972 g/L. H. on this basis. The yield of succinic acid was 76.5 g/L. The yield and yield of succinic acid were 1.063 g/L. H and 76.5%. respectively. Finally, succinic acid in the fermentation liquid was separated by calcium salt method. The large bulk material was removed by filtration, and most of the bacteria were removed through ceramic membrane filtration. Succinic acid was separated and extracted by calcium salt precipitation, decolorization of activated carbon and ion exchange, and the calcification process was optimized. The optimum calcification reaction condition was 30% calcium chloride solution of equal volume at 80 C under the condition of adding 3. 0 min. finally obtained succinic acid crystallization 99.6 g, succinic acid yield was 56.8%.
【学位授予单位】：湖北工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ921;Q933

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