论文总字数：25351字

摘 要

2,3-丁二醇是重要的化工产品和液体燃料，广泛应用于化工、食品、燃料及航空航天等领域，具有生物降解性。本文旨在提高Paenibacillu spolymyxa ZJ-9发酵制备R,R-2,3-丁二醇产量，具体研究内容如下：

将来源于Candida boidinii中的fdh基因与来源于Bacillus subtilis 168的 P43启动子进行重叠PCR后构建至 pMA5 质粒，获得重组表达载体pMA5-fdh，成功将重组表达载体pMA5-fdh电转入P. polymyxa ZJ-9中。在7.5 L罐中考察了野生菌P. polymyxa ZJ-9与重组菌P. polymyxa XG-1分批发酵过程中菌体生长，FDH酶活，胞内NADH/NAD 以及合成 2,3-BD 的过程中代谢产物的变化。结果表明，发酵初期野生菌生长比重组菌生长较快，发酵中后期野生菌与重组菌生长相当，重组菌P. polymyxa XG-1在24 h时，重组菌的甲酸脱氢酶表达量最高，达到1.73 U/mg，而野生菌酶活很低，发酵过程中重组菌NADH高于野生菌，而NAD 低于野生菌，发酵到44 h时，R,R-2,3-丁二醇产量达到36.8 g/L，与野生菌发酵结果相比，产量提高了10.2%，重组菌乙偶姻的产量降低为0.88 g/L，乳酸以及乙醇含量都有所提高，重组菌的甲酸含量相对较低，在30 h以后，重组菌发酵液中甲酸含量维持在0.6 g/L左右。

关键词：P. polymyxa ZJ-9 R,R-2,3-丁二醇 甲酸脱氢酶 丁二酮还原酶

ABSTRACT

2,3-butanediol (2,3-BD) is an important chemical material and liquid fuel that is widely applied in chemical industry, food, fuels and aerospace field because of its biodegradability. In order to increase the yield and optical purity of R,R-2,3-BD, the researches were made as follows.

The gene fdh from Candida boidinii and P43 promoter from Bacillus subtilis 168 were constructed after overlapping PCR to pMA5 plasmid, recombinant expression vector pMA5-fdh, which was successfully transferred into P. polymyxa ZJ-9. In 7.5 L fermentor was studied in a batch fermentation process of cell growth between the wild fungus P. polymyxa ZJ-9 and recombinant bacteria P. polymyxa XG-1, FDH enzyme activity, the metabolite changes in intracellular NADH/NAD and metabolites of 2,3-BD synthesis. The results show that, at the initial stage of fermentation wild mushroom growth was faster than the growth of recombinant bacteria, and in the late of fermentation wild fungi and recombinant bacteria grow quite, recombinant P. polymyxa XG-1 at 24 h, formate dehydrogenase expression was the highest, reached 1.73 U/mg, while the wild mushroom enzyme activity is very low, the fermentation process of recombinant NADH was higher than that of the wild bacteria, and NAD was lower than that of wild strain. And fermentation to 44 h, R,R-2,3-butanediol production reached 36.8 g/L, compared with the results of wild fungi fermentation, the yield increased by 10.2%, acetoin yield is reduced to 0.88 g/L, lactic acid and ethanol content was increased, the recombinant bacteria formic acid content is relatively low, in the after 30 h, formic acid in the fermentation liquid of recombinant bacteria maintained at about 0.6 g/L.

KEYWORDS P.polymyxaZJ-9; R, R-2,3-butanediol; Formate dehydrogenase; Diacetyl reductase

目录

摘 要 I

ABSTRACT II

第一章 文献综述 1

1.1 引言 1

1.2 2,3-丁二醇 1

1.2.1 2,3-丁二醇的分类 1

1.2.2 2,3-丁二醇的主要应用 1

1.2.3 2,3-丁二醇的市场 1

1.2.4 2,3-丁二醇的生产方法 2

1.2.5 2,3-丁二醇的生产菌种 2

1.3 辅酶再生 3

1.3.1 辅酶再生的意义 3

1.3.2 辅酶再生的原理及方法 3

1.4 甲酸脱氢酶 4

1.4.1 甲酸脱氢酶的概念 4

1.4.2 研究甲酸脱氢酶的意义 4

1.5菊芋 5

1.5.1 菊芋概述 5

1.5.2 菊芋的应用 5

1.5.3 利用菊芋作为碳源的意义 6

1.5.4 菊芋的研究进展 6

1.6 论文的意义及内容 7

第二章 实验材料与方法 8

2.1 实验材料 8

2.1.1 菌种与质粒 8

2.1.2 实验仪器及试剂 8

2.1.3 培养基及培养条件 9

2.2 实验方法 10

2.2.1 转化方法 10

2.2.2 含fdh基因表达载体的构建 10

2.3 分析方法 13

2.3.1 细胞生长的测定 13

2.3.2 酶活力的测定 13

2.3.3 细胞内NADH/NAD 含量的测定 13

2.3.4 R,R-2,3-丁二醇及副产物的含量测定 14

2.3.5 CER含量测定 14

第三章 结果与讨论 16

3.1 P.polymyxaZJ-9的生长曲线 16

3.2 标准曲线的绘制 16

3.3 P43启动子和fdh基因的PCR扩增 17

3.4 重叠PCR片段 17

3.5 克隆载体pMD-pf的构建及鉴定 18

3.6 pMA5-pf重组质粒的构建及验证 18

3.7 pMA5-P43-fdh转化P. polymyxa ZJ-9 18

3.8 FDH对2,3-丁二醇发酵菌体生长过程的影响 19

3.9 发酵过程中FDH酶活的变化 20

3.10 FDH对2,3-丁二醇发酵菌体中NADH及NAD 的影响 21

3.11 FDH对2,3-丁二醇发酵过程代谢流的影响 23

3.12 本章小结 25

第四章 结论与展望 26

4.1 结论 26

4.2 展望 26

参考文献 28

致 谢 30

第一章 文献综述

1.1 引言

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