论文总字数：17063字

摘 要

高密度发酵技术是指在一定的培养体系中，通过改进培养方式和培养条件，提高菌体的发酵密度的培养技术，一般在液体培养基中的细胞密度超过常规培养的10倍以上。与其他发酵技术相比，可以减少培养体积，降低设备投资，降低生物量的分离费用，提高产物产量，提高生产效率，是目前广泛应用的培养技术。

本实验通过单因素实验，在摇瓶体系中，通过在不同的发酵培养基中培养目的菌株，根据菌体浓度高低和酶活，优化发酵培养基的碳源和氮源。在发酵过程中，采用双阶段控氧。前期，通过提高转速和加大通气量来维持溶氧，当转速和通气量达到上限后，通入空气和纯氧混合气体以维持溶氧在20%到30%，实现大肠杆菌的高密度发酵。此供氧方法得到的OD达65，酶活力达1.5×10⁴ U/mL，较只通空气的普通发酵分别提高了1.3倍和0.5倍。

本实验所提供优化后的发酵培养基，有利于菌体的生长。采用的双阶段供氧的方法，维持溶氧，提高了大肠杆菌的生物量，提升了海藻糖合成酶的表达水平，降低了生产成本，提高了生产效率。

关键词： 高密度发酵 溶氧 发酵培养基

Production of trehalose synthase by high density fermentation of recombinant Escherichia coli

ABSTRACT

High density fermentation technology that is changing culturing methods and conditions to improve the fermentation density of the culture technology in a certain culturing system. The cell density in liquid medium was 10 times more than that ordinary medium. Compared with other fermentation technology, it can reduce the culture volume, reduce the equipment investment, reduce the cost of separating biomass, increase the output of the product, and improve the production efficiency. High density fermentation technology is widely used at present.

This experiment cultures strains in different fermentation medium in the shake flask system with the single factor experiment. We culture the strain in different fermentation medium and compare the results to optimize carbon source and nitrogen source in the fermentation medium. This experiment adopts double-phase dissolved Oxygen control during the fermentation process. In the early stage of the fermentation, we improve the speed and increase ventilation to maintain dissolved oxygen. When the rotation speed and ventilation reach ceiling, we use the mixture of the air and pure oxygen gas to keep the dissolved oxygen between 20% and 30%, realizing high density fermentation of Escherichia coli. This method make the optical density (OD) reach 65and the enzyme activity reach 1.5×10⁴ U/mL, which increased by 1.3 times and 0.5 times. Compared with the ordinary fermentation method.

This study provides an optimized fermentation culture medium that is beneficial to the growth of strains. This study adopts double stage dissolved oxygen control, increasing the density of Escherichia coli. The results showed that the expression level of trehalose synthase was improved. The cost of the production is reduced and the efficiency of production is improved.

KEYWORDS: High density fermentation Dissolved oxygen Fermentation culture medium

目录

摘 要 I

ABSTRACT II

第一章 文献综述 1

1.1海藻糖概述 1

1.1.1 海藻糖简介 1

1.1.2海藻糖制备的研究 2

1.1.3 海藻糖的应用 2

1.2大肠杆菌高密度发酵技术 3

1.2.1 宿主菌类型 3

1.2.2 培养基组成 3

1.2.3 接种量 4

1.2.4 培养条件 4

1.2.5 补料方法 5

1.2.6发酵终点的判断 6

1.3.酶的提取检测 6

1.3.1 酶的提取 6

1.3.2 酶的检测 6

1.4 本课题研究的目的和意义 7

第二章 实验材料与方法 7

2.1实验材料 8

2.1.1 实验试剂 8

2.1.2 实验仪器 8

2.1.3 培养基 9

2.1.4 实验菌种 9

2.2 实验方法 9

2.2.1 大肠杆菌高密度发酵培养 9

2.2.2 PH-stat补料 10

2.2.3 高密度发酵的溶氧控制 10

2.2.4 海藻糖合成酶的获取与检测 10

2.2.5 生物量（OD₆₀₀）的测定 10

2.2.6 酶活力计算 10

第三章 结果与分析 12

3.1发酵培养基碳源的优化 12

3.2发酵培养基氮源的优化 13

3.3 海藻糖标准曲线的制作 14

3.4 普通供氧发酵与双阶段供氧发酵的比较 17

第四章 结论与展望 19

4.1结论 19

4.2展望 19

参考文献 20

致谢 21

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