论文总字数：21297字

摘 要

γ-聚谷氨酸是由L-谷氨酸（L-Glu）及D-谷氨酸（D-Glu）的γ-羧基与氨基脱水缩合构成γ-酰胺键而形成的高分子化合物，是工业上用途广泛的材料，可以应用于包括农业、食品、化妆品、医药等众多领域。γ-聚谷氨酸的分子量在50 kDa到2000 kDa之间。而γ-聚谷氨酸的用途主要取决于它的分子量，它的分子量大小又取决于它的生产方式。目前主流的大规模工业化生产γ-聚谷氨酸的方法是微生物发酵法。但是作为典型的高黏好氧发酵体系，在发酵后期，由于产物的大量积累造成的黏度升高、溶氧降低会给发酵过程带来困难。传统的解决方法是提高搅拌速率，但是这样会造成γ-聚谷氨酸的分子量的下降。同时，发酵过程中，菌株的底物利用率不足，高浓度底物的耐受度不足也是亟待解决的问题。本实验尝试了通过适应性进化的策略驯化出能够耐受低溶氧的环境和耐受高葡萄糖浓度的γ-聚谷氨酸高产枯草芽孢杆菌菌株。

本实验从枯草芽孢杆菌NX-2菌株出发，通过低溶氧与高葡萄糖浓度环境对菌株进行适应性进化实验，获得两株γ-聚谷氨酸高产菌株，通过HPLC实验验证产量，其中LDOH菌株的γ-聚谷氨酸产量已经超越了NX-2起始菌株的6.97%，HG菌株的产量超过了起始菌株的5.78%。同时，以OD₆₀₀为发酵过程中生物量的参照，证实生物量与起始菌株相比也维持了稳定。从底物消耗率方面，两株菌株的底物利用率与起始菌株相比都有了提升。两株菌的葡萄糖利用率与起始菌株相比得到了大大的提升，在发酵后期（36 h）取样，LDOH菌株的葡萄糖消耗率相比NX-2提升了46.03%，而HG菌株的糖耗相比NX-2提升了56.38%。而二者对谷氨酸钠的消耗也有了一定程度的提升，LDOH菌株的谷氨酸钠消耗量相较起始菌株增加了5.90%，而HG菌株相较起始菌株增加了6.64%。

关键词：γ-聚谷氨酸 枯草芽孢杆菌 适应性进化 发酵工

Adaptive evolutionary strategy to enhance the synthesis of γ-polyglutamic acid from Bacillus subtilis

Abstract

γ-polyglutamic acid is a polymer compound formed by dehydration condensation of a γ-carboxyl group of L-glutamic acid (L-Glu) and D-glutamic acid (D-Glu) with an amino group to form a γ-amide bond. As a water-soluble biopolymer, γ-polyglutamic acid can be biodegraded, and also has metal chelation and biocompatibility characteristics. Therefore, it has become an industrially versatile material and can be applied. Including agriculture, food, cosmetics, medicine and many other fields. The molecular weight of γ-polyglutamic acid is between 50 kDa and 2000 kDa. The use of γ-polyglutamic acid depends mainly on its molecular weight, and its molecular weight depends on its production mode. At present, the mainstream large-scale industrial production method of γ-polyglutamic acid is a microbial fermentation method. However, as a typical high-viscosity aerobic fermentation system, in the late stage of fermentation, the viscosity increased due to the large accumulation of products, and the dissolved oxygen will bring difficulties to the fermentation process. The traditional solution is to increase the rate of agitation, but this will result in a decrease in the molecular weight of γ-polyglutamic acid. At the same time, during the fermentation process, the substrate utilization rate of the strain is insufficient, and the tolerance of the high concentration substrate is also an urgent problem to be solved. This experiment attempted to domesticate a high-yield γ-polyglutamic acid-producing Bacillus subtilis strain that was able to tolerate low dissolved oxygen and tolerate high glucose concentrations through an adaptive evolutionary strategy.

In this experiment, Bacillus subtilis NX-2 strain was used to carry out adaptive evolution experiments on strains with low dissolved oxygen and high glucose concentration environment. Two strains of γ-polyglutamic acid high-yield strains were obtained, and the yield was verified by HPLC experiments. The yield of γ-polyglutamic acid has exceeded 6.97% of the NX-2 starting strain, and the yield of HG strain exceeded 5.78% of the starting strain. At the same time, OD₆₀₀ was used as a reference for biomass in the fermentation process, and it was confirmed that the biomass was also stable compared with the starting strain. In terms of substrate consumption rate, the substrate utilization rate of the two strains was improved compared with the starting strain. The glucose utilization rate of the two strains was greatly improved compared with the original strain. At the late stage of fermentation (36 h), the glucose consumption rate of LDOH strain was increased by 46.03% compared with NX-2, and the sugar consumption phase of HG strain was increased. It is 56.38% higher than NX-2. The consumption of sodium glutamate was also improved to some extent. The consumption of sodium glutamate in LDOH strain was 5.90% higher than that in the original strain, while the HG strain was increased by 6.64% compared with the original strain.

Keywords：γ-polyglutamic acid；Bacillus subtilis；adaptive evolution；fermentation engineering

目 录

摘要 Ⅰ

Abstract Ⅱ

第一章 文献综述 1

1.1适应性进化 2

1.2 γ-聚谷氨酸 2

1.2.1 γ-聚谷氨酸的物理化学性质 2

1.2.2 γ-聚谷氨酸的主要生产方式 3

1.2.3 γ-聚谷氨酸的应用 4

1.3 研究内容 6

第二章 菌株适应性进化及筛选 7

2.1前言 7

2.2 低溶氧适应性进化 7

2.2.1 菌株 7

2.2.2 培养基 7

2.2.3 实验试剂 8

2.2.4 实验仪器 8

2.2.5 实验方法 9

2.2.6 分析方法 10

2.3 高浓度底物适应性进化 11

2.3.1 菌株 11

2.3.2培养基 11

2.3.3 实验试剂 12

2.3.4 实验仪器 12

2.3.5 实验方法 12

2.3.6 分析方法 13

第三章 结果与讨论 14

3.1 表观菌落变化 14

图1-1 NX-2菌株与驯化菌株ALE12平板培养菌落形态对比 14

3.2 发酵产量分析 15

表2-1：低溶氧驯化菌种在不同时期的发酵产量 16

表2-2：高糖驯化菌种在不同时期的发酵产量 16

3.3细胞生长状况分析 16

图3-1：各代ALE-LDOH菌株生长过程中的OD600变化趋势图 17

图3-2：各代ALE-HG菌株生长过程中的OD600变化趋势图 18

3.4驯化菌株发酵过程中的底物消耗情况分析 18

图4-1：ALE-HG菌株发酵过程中的葡萄糖余量图 19

图5：第二十代菌株与起始菌株发酵过程中的谷氨酸余量图 20

第四章 结论与展望 21

4.1 结论 21

4.2 展望 21

参考文献 22

致谢 25

第一章 文献综述

1.1 适应性进化

实验室适应性进化（ALE）在生物工程学科的研究中是一种极为重要的研究方法。其中心思想发端于19世纪中叶的达尔文的进化论及赫胥黎发展的现代综合进化论，将这两种思想应用于现代的全面、系统的微生物工程中，便诞生了适应性进化的实验方法。微生物具有根据外界快速改变其遗传物质及表征以适应所给环境的能力。利用微生物的这种特点，我们可以给予微生物一定的生长压力，经过长期的传代、筛选，从而获得适应了某种特定环境的菌株，并且能相应的提高某种特定次级代谢产物的得率。用进化论思想解决生物工程中的复杂工程问题已经成为一种备受关注的思路。

生长压力的选择对于适应性进化的成功与否至关重要。生长压力大体上可以分为两类：营养压力与环境压力。营养压力是指来自于对培养基成分中碳源、氮源等营养成分做出相较于菌株自身最适于生长或代谢的改变而带来的生长压力，例如高糖、高无机盐等等。红球菌（Rhodococcus opacus）只能使用葡萄糖和木糖作为底物来产生脂质，而Kurosawa等人选育了一株红球菌菌株，通过适应性进化后使其能够在含100 g/L甘油的培养基的高甘油浓度下进行高细胞密度培养^[1]。与红球菌类似，在对于酿酒酵母（Saccharomyces cerevisiae）的研究中也广泛应用了营养压力驱动的适应性进化策略^[2]。对葡萄糖限制恒化器的早期研究表明，葡萄糖限制选择导致生物量增加和发酵能力降低。

1.2 γ-聚谷氨酸

1.2.1 γ-聚谷氨酸的物理化学性质

γ-聚谷氨酸（γ-PGA）是一种由L-谷氨酸（L-Glu）及D-谷氨酸（D-Glu）的γ-羧基与氨基脱水缩合构成γ-酰胺键而形成的多肽高分子化合物^[3]。其结构式如图所示。

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