【作者】 邵敬伟；

【导师】 刘长江；

【作者基本信息】 沈阳农业大学 ， 食品科学， 2004， 博士

【摘要】 1,3-丙二醇（1,3-PD）是一种重要的化工原料,具有许多优良特性和很好的应用前景。目前生物转化法生产1,3-PD 以其利用再生资源、对环境污染小等特点越来越受到人们的重视。而甘油脱水酶（glycerol dehydratase,GDHt）是1,3-PD 生物合成途径中涉及到的第一个酶,也是控制甘油分解和产生1,3-PD 的关键性限速酶,对1,3-PD 的转化速率起着至关重要的作用,因此甘油脱水酶的研究对于我国1,3-PD 的进一步开发利用具有重要的实际意义。本论文的研究目的是通过研究肺炎克氏杆菌发酵法合成1,3-PD 的情况和肺炎克氏杆菌菌株中甘油脱水酶的基因从而实现用基因工程手段实现1,3-PD 的生物合成,提高1,3-PD 的产量,并可以填补国内重组基因工程菌生产1,3-PD 基础研究的空白。本文主要研究了肺炎克氏杆菌发酵甘油生产1,3-PD 的情况并在GDHt基因的克隆、测序、鉴定以及其表达载体的构建方面开展了一些工作。首先对肺炎克氏杆菌A.S.1.1736 发酵甘油生产1,3-PD 的发酵培养基和发酵条件进行了探索。在上述实验基础上,在国内首次利用PCR 从肺炎克氏杆菌基因组DNA 中扩增出编码甘油脱水酶三个亚基的基因片段,分析其核苷酸和氨基酸序列特征,并且利用pMAL-c2X 和pGEX-4T-1 载体分别构建了三种基因的单独表达载体和三种基因的串联表达载体,共计九种克隆及表达载体。我们取得的主要结果如下:对Klebsiella pneumoniae A.S.1.1736 发酵甘油生成1,3-PD 的发酵培养基和发酵条件进行了探索。考察了C、N、P 和Fe²⁺等单因子对发酵的影响,确定了最佳发酵培养基的配方为甘油60g/L,NH4Cl 1.8g/L,KH2PO4 1.6g/L,Fe2+ 0.03‰;通过正交实验确定了1,3-PD 发酵条件:最适pH 值、温度、时间和接种量分别为7.0、37℃、40h、8%;在最适发酵培养基和最适发酵条件下,1,3-PD 最高产量可达38.62g/L。通过PCR 方法获得了克氏杆菌中编码甘油脱水酶三个亚基（α、β、γ）的基因片段。并进行PCR 产物直接测序和克隆至pMD18-T 载体测序。经核苷酸序列分析证实:大基因开放阅读框共1668 个核苷酸,是以ATG 起始密码子开始,TGA 终止密码子结束,共编码555 个氨基酸,G+C 含量为60.54%;更多还原

【Abstract】 1,3-Propanediol is an important substance in chemical industries. It has a lotof good character and wide application future. It has been paid more and moreattention for its utilization rebirth resource and little pollution to the environmentin the bioconversion of 1,3-PD. The glycerol dehydratase (GDHt) is the firstenzyme involved in the process of bioconversion of 1,3-PD and catalyzes therate-limiting step in the anaerobic conversion of glycerol to 1,3-PD, it has themost important effect to the conversion speed of 1,3-PD. So the research ofglycerol dehydratase has important actual meaning to home further developmentand utilization of 1,3-PD. The aim of our study is to initiate the reseach by therecombinant microorganism in the bioconversion of 1,3-PD and improve theyield of 1,3-PD by the search of microbial fermentation production of 1,3-PD byKlebsiella pneumoniae and the study of genes encoding glycerol dehydratase.In this experiment, the microbial fermentation production of 1,3-PD by K.pneumoniae was studied and gene cloning, sequence analysis and construction ofexpression vector of GDHt were first described in our country. Firstly, theoptimum fermentation culture medium and conditions of K. pneumoniae weredetermined. Based on what mentioned above, the genes encoding GDHt were clonedby using PCR method. The sequence analysis and construction of expression vectorfrom pMAL-c2X and pGEX-4T-1 vector in E.coli wereaccomplished respectively.The major results are as the following:The optimum fermentation culture and conditions in the microbialfermentation production of 1,3-PD by K. pneumoniae were determined. Effects ofC、N、P and Fe2+ was studied. The optimum culture medium is: glycerol 60g/L,NH4Cl 1.8g/L,KH2PO4 1.6g/L,Fe2+ 0.03‰;Through orthogonal experiment,theoptimum culture conditions are:pH 7.0、temperature 37℃、culture time 40h、inoculum size 8%;Under the optimum conditions: the maximal 1,3-PD yield was38.62g/L.The genes encoding glycerol dehydratase of K. pneumoniae were clonedby using PCR method respectively. The PCR products were sequenced directlyand cloned to pMD18-T and identified by sequencing respectively. The sequenceresult of the amplified DNA fragments shows: Large gene fragment was 1668base pairs encoding 555 amino acid residues, it included an initiation codon(ATG)and a stop condon(TGA), the G+C content was 60.54%;The middle gene更多还原