论文总字数：25539字

摘 要

丁二酸是工业上一种重要的四碳化合物，可作为有机合成原材料、中间产物或化学品，广泛应用于医药、食品、农业、精细化工等领域。研究玉米芯发酵生产琥珀酸，不仅可以降低成本，促进农林废弃物利用，同时也可避免“与人争粮，与粮争地”等问题的产生，具有重要的实际应用意义。微生物合成目的产物主要存在三个问题：目的产物产量不高、产品转化效率不高、生产强度不大。本论文针对谷氨酸棒杆菌厌氧合成丁二酸过程中存在的具体问题展开如下研究：

谷氨酸棒杆菌胞内高能荷水平别构抑制糖酵解途径中关键酶活性，降低糖酵解速率，因此可以通过降低胞内能荷水平以加快菌株糖酵解速率。通过定点突变H -ATPase基因中一个碱基，降低其活性。突变株C. glutamicum NC-3b-6胞内ATP水平下降43.7%时表现出较高的葡萄糖消耗速率。糖酵解途径中关键酶活性有所提高，表明胞内能荷水平的降低解除了对糖酵解途径关键酶的抑制。5 L发酵罐厌氧补料发酵48 h，重组菌株C. glutamicum NC-3b-6合成丁二酸产量达950±5.3 mM，生产强度为19.8±2.1 mM/h，丁二酸转化率为1.44±0.3 mol/mol。

关键词：丁二酸 谷氨酸棒杆菌 能荷水平 糖酵解速率 转化率

ABSTRACT

Succinic acid, an important industrial carbon compounds, is widely used in medicine, food, agriculture, fine chemicals and other fields as the organic synthesis of raw materials, intermediate products or chemicals. The production of the succinic acid by corn cob hydrolyzate could not only reduce costs, promote agricultural and forestry wastes utilization, but avoid issues on competing with food, fighting with land, which has important practical applications of significance. There are three main issues in the microbial of desired products: not high production of chemicals, not high yield and not high productivity. There are two main reasons: the insufficient ability of cell synthesis and the poor conditions of fermentation process. This thesis aimed to investigate the ability of Corynebacterium glutamicum for succinic acid production. The main contents and conclusions were described as following:

Dutch energy level within glutamicum intracellular allosteric inhibition of the glycolytic pathway key enzyme activity, reduce the rate of glycolysis,So，the level of ATP was decreased to increase the rate of glycolysis. A point mutation in H -ATPase resulted in a decreased ATP level. The activity of key enzymes in glycolysis were increased by the decreased ATP level. In C. glutamicum NC-3b-6, the production of succinic acid was 950±5.3 mM, the productive of succinic acid was 19.8±2.1 mM/h, the yield of succinic acid was 1.44±0.3 mol/mol.

KEYWORDS: Succinic acid; Corynebacterium glutamicum; ATP level；Glycolytic rate；Conversion rates

目录

摘 要 I

ABSTRACT II

目录 Ⅲ

第一章 文献综述 1

1.1 丁二酸的性质、用途及市场前景 1

1.1.1 丁二酸的性质 1

1.1.2 丁二酸的用途 1

1.1.3 丁二酸的市场前景 1

1.2丁二酸的生产方法 2

1.2.1 化学法合成丁二酸 2

1.2.2 生物转化法合成丁二酸 3

1.2.3 微生物发酵法 3

1.3 发酵法生产丁二酸的菌株及其研究进展 3

1.3.1产丁二酸的菌株种类及其产量比较 3

1.4 能量代谢对糖酵解水平的调控及应用 5

1.5 本实验研究的意义和主要内容 6

第二章 材料与方法 6

2.1 实验材料与仪器 7

2.1.1 菌株与质粒 7

2.1.2实验试剂、仪器、工具酶、分子量标准及试剂盒 7

2.1.2.1 工具酶、分子量标准及试剂盒 7

2.1.2.2 实验试剂 8

2.1.2.3 实验仪器 10

2.2 培养基及培养条件 11

2.3 菌体生长培养方法 12

2.4 感受态制备及转化方法 12

2.4.1大肠杆菌感受态制备及转化 12

2.4.2谷氨酸棒杆菌电转感受态细胞制备及电转化 13

2.5 糖酵解途径关键酶和H -ATPase活性的测定 13

2.6 重组质粒pK18mobsacB-atpG*的构建及验证 14

2.6.1 目的片段atpG*的获得 14

2.6.2 重组质粒pK18mobsacB- atpG*的构建及验证 14

2.6.3 重组菌株C. glutamicum NC-3b-6的构建及验证 15

第三章 实验结果与讨论 15

3.1 重组菌株C. glutamicum NC-3b-6的构建及验证 16

3.1.1重组质粒pK18mobsacB- atpG*的构建及验证 16

3.1.2 重组菌株C. glutamicum NC-3b-6的构建及鉴定 17

3.2 重组菌株C. glutamicum NC-3b-6 胞内辅因子及ATP的测定 17

3.3 H -ATPase活性降低对C. glutamicum NC-3b-5厌氧发酵的影响 18

第四章 结论与展望 19

4.1 结论 20

4.2 展望 20

参考文献 20

第一章 文献综述

1.1 丁二酸的性质、用途及市场前景

1.1.1 丁二酸的性质

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