论文总字数：16054字

摘 要

L- 鸟氨酸是人体代谢中必不可少的中间代谢产物，一种非蛋白质氨基酸,以游离的形式存在于组织和细胞中,具有重要的生理活性和功能,有着很强的研究开发价值。作为尿素生成和多胺代谢的中间产物,具有解氨毒及治疗肝病等多种生物功能,在医疗、保健、食品等领域有重要作用使其愈来愈广泛的应用到食品工业和医药工业中这都将给L-鸟氨酸产品提供巨大的市场空间。化学法产生的鸟氨酸是外消旋混合物,且反应步骤多,收率不高;水解精氨酸法生产L -鸟氨酸受制于原料精氨酸;发酵法制备L -鸟氨酸原料低廉,通过改造菌种和改进发酵工艺可以提高鸟氨酸的发酵水平,因此发酵法制备鸟氨酸有着长远的经济意义。

谷氨酸棒杆菌是多种重要氨基酸的工业生产菌株,也是鸟氨酸的生产菌株。根据生产菌株的代谢特性，分别考察培养基的碳氮源组成、pH、生物素含量、初糖浓度对发酵过程的影响，结合补料发酵手段，提高L-鸟氨酸的生产速率和转化率，初步得到以下结论：

当种子培养基中尿素含量为2.5g/L时，种子生长和L-鸟氨酸生产性能较好。种子培养基 pH 值过低或过高都不利于菌体生长， pH 7.5时具有较好的鸟氨酸生产性能。

生物素含量的不同对菌体生长和发酵产酸都有影响，而控制生物素含量在80μg/L对菌种生长和发酵产酸最为有利。当初始葡萄糖浓度浓度高于90 g/L，菌体和产物合成量明显减少，可能存在底物抑制。在此条件下平均耗糖速率为1.17 g/L/h，产L-鸟氨酸22.67 g/L，对糖收率为27%。

通过补糖分批发酵，可明显提高L-鸟氨酸生产性能，可将120g/L的葡萄糖完全消耗， L-鸟氨酸产量和生产速率分别达到33.25 g/L、0.46g/L/h，比120g/L初糖的分批发酵分别提高了117%、116%，转化率为27.8%。

关键词：L-鸟氨酸 谷氨酸棒杆菌 调控 发酵优化

ABSTRACT

L- ornithine is essential to human metabolism intermediate metabolite, a non-protein amino acid, in free form in the tissues and cells has important physiological activities and functions, has a strong research and development value. As urea production and intermediate polyamine metabolism has ammonia poisoning and treatment of liver disease and other biological functions, in medical and health care, food and other fields have an important role to make it more widely applied to the food industry and the pharmaceutical industry this will give L- ornithine products provide a huge market space. Chemically produced racemic mixture is ornithine, and multi-step reaction, the yield is not high; arginine hydrolysis produced L - arginine, ornithine subject material; Fermentation Preparation L - ornithine material low, through the transformation of bacteria and improve the fermentation process can increase the level of fermentation of ornithine, ornithine therefore prepared fermentation has long-term economic sense.

Glutamicum strain is a variety of important industrial strain of amino acids production. According to the metabolic characteristics of the strains were investigated fermentation medium composed of carbon and nitrogen sources, initial glucose concentration, pH, biotin content, the impact of initial glucose concentration in the fermentation process, combined with batch fermentation methods, improve L- ornithine production rate and conversion. The main conclusion was as follows:

When the seed medium urea content of 2.5g / L, better growth and seed production performance L- ornithine. The seed medium pH value is too low or too high is not conducive to bacterial growth, pH 7.5 ornithine having good performance.

Cell growth and L- ornithine production were affected by biotin, and the biotin content 80 μg/L was favorable for the growth of bacteria and fermentation. When the initial concentration of glucose concentration was higher than 90 g/L, cell and product synthesis was significantly reduced, there may be substrate inhibition. Under these conditions, the average sugar consumption rate was 1.17 g/L/h, L- ornithine 22.67 g/L was produced, sugar yield was 27%.

L- ornithine production was improved significantly by fed-batch fermentation. 120 g/L glucose was completely consumed, L-ornithine yield and productivity reached 33.25 g/L and 0.46 g/L/ h, which were increased by 117%, 116% compared with batch fermentation with 120 g/L initial glucose, and the yield was 27.8%.

Key words: L-onrithine；Corynebacterium glutamicum； Regulate and Control ；Fermentation optimization

目录

摘要 I

ABSTRACT II

目录 V

第一章 文献综述 1

1.1 鸟氨酸(Ornithine)简介 1

1.1.1 鸟氨酸结构和性质 1

1.1.2 L-鸟氨酸的生物功能 1

1.2 L-鸟氨酸的制备方法 2

1.3 L-鸟氨酸的生物合成途径 2

1.4 L-鸟氨酸发酵条件的研究概况 3

1.4.1 培养基组成成分对产酸的影响 3

1.4.2 培养条件对产酸的影响 3

1.5 本文的研究目标及内容 4

第二章 实验材料与方法 5

2.1 实验试剂 5

2.1.1 实验试剂 5

2.1.2 仪器 6

2.2 菌种 7

2.3 培养基 7

2.3.1 LB固体培养基 7

2.3.2 种子培养基 7

2.3.3 发酵培养基 7

2.4 培养方法 7

2.4.1 固体平板培养（LB培养基） 7

2.4.2 种子培养 7

2.4.3 发酵培养 7

2.5 分析方法 8

2.5.1 菌体生长测定 8

2.5.2 发酵液中残糖测定 8

2.5.3 发酵液中L-鸟氨酸的测定 8

第三章 结果与讨论 10

3.1 L-鸟氨酸标准曲线的绘制 10

3.2 种子培养的初步考察 11

3.2.1 培养基氮碳比对种子培养及发酵性能影响 11

3.2.2 初始pH 对种子培养及发酵性能的影响 11

3.2.3 生物素添加量对发酵产鸟氨酸的影响 12

3.2.4 初糖浓度对发酵产鸟氨酸的影响 13

3.2.5 分批补糖对发酵产鸟氨酸性能的影响 14

第四章 结论和展望 16

4.1 结论 16

4.2 展望 16

参考文献 17

致谢 19

第一章 文献综述

1.1 鸟氨酸(Ornithine)简介

1.1.1 鸟氨酸结构和性质

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