论文总字数：20653字

摘 要

天然产物纽莫康定B₀是首个半合成棘白菌素类抗真菌药物卡泊芬净的前体物质，可通过抑制β-1,3-D-葡聚糖合成酶活性而对真菌细胞壁造成伤害，从而达到抗真菌感染的效果。纽莫康定B₀由丝状真菌Glarea lozoyensis发酵生产，是一种在发酵过程中主要积聚在菌丝体内的疏水性次级代谢产物，所以发酵后期会出现产物反馈抑制。

前期研究中发现添加表面活性剂SDS可以提高纽莫康定B₀的产量。在此基础上，本论文研究了SDS促进纽莫康定B₀生产的机制。主要研究结果如下：在纽莫康定发酵过程中添加1.0 g/L SDS，胞外纽莫康定B₀产量比对照组提高了153.78%。相比于对照组，实验组中细胞内饱和脂肪酸C16:0和C18:0分别减少了44.94%和55.02%，不饱和脂肪酸C18:1和C18:2的比例分别增长了133%和72%。采用NPN法，检测细胞膜的通透性，发现实验组的荧光强度增加了2倍，表明相对于对照组，实验组中细胞膜通透性增加。同时，相比于对照组，实验组中的菌丝和颗粒的直径分别减少了14%和7%，菌丝表面产生致密的褶皱，菌丝粗细不均，甚至菌丝表面出现破损。因此，添加SDS可以提高生产菌株的细胞膜通透性，将胞内纽莫康定B₀不断富集到胞外萃取剂中，促进胞内纽莫康定B₀合成。

关键字：Glarea lozoyensis 纽莫康定B₀ 发酵 细胞膜通透性

ABSTRACT

The natural product, pneumocandin B₀, is the precursor of the first semi-synthetic echinocandin antifungal drug caspofungin, which can cause damage to the fungal cell wall by inhibiting the activity of β-1,3-D-glucan synthase to achieve antifungal infection effects. Pneumocandin B₀ is produced by fermentation of the filamentous fungus Glarea lozoyensis, which is a hydrophobic secondary metabolite mainly accumulated in the mycelium during fermentation, so it is easy to be product feedback inhibition in the late stage of fermentation.

Previous studies have found that the addition of surfactant SDS can increase the yield of pneumocandin B₀. On this basis, this paper studied the mechanism of SDS to promote the increase of pneumocandin B₀. The main results are as follows: When 1.0 g/L SDS was added during the fermentation of pneumocandin B₀, the yield of extracellular pneumocandin B₀ was increased by 153.78% compared with the control group. The proportions of intracellular saturated fatty acids(C16:0 and C18:0) in the experimental group decreased by 44.94% and 55.02% respectively and the proportions of unsaturated fatty acids (C18:1 and C18:2) increased by 133% and 72% respectively relative to the control group.. The cell membrane permeability was measured by the NPN method. The fluorescence intensity of the experimental group increased by two times, which indicate that the cell membrane permeability was increased in the experimental group relative to the control group. At the same time, the diameters of the hyphae and the particles in the experimental group were reduced by 14% and 7% respectively.The surface of the hyphae produced dense wrinkles and thickness was uneven. Even the surface of the hyphae was damaged. Therefore, we know it can improve the cell membrane permeability of the production strain by the adding of SDS which continuously enrich the intracellular pneumocandin B₀ into the extracellular extractant to promote the intracellular pneumocandin B₀ synthesis.

Key Words: Glarea lozoyensis; Pneumocandin B₀; Fermentation; Cell membrane permeability

目录

摘要 I

ABSTRACT II

目录 3

第一章 文献综述 5

1.1 纽莫康定B₀简介 5

1.2 纽莫康定类化合物理化性质和结构 5

1.3 纽莫康定B₀合成机制 6

1.4 G. lozoyensis产纽莫康定B₀发酵工艺进展 8

1.5 表面活性剂应用简介 9

1.6 研究思路与意义 9

第二章 实验材料与方法 11

2.1 菌种 11

2.2 主要仪器和试剂 11

2.3 培养基组成 13

2.4 培养方式 13

2.5 检测方法 13

2.5.1 细胞干重测定 13

2.5.2 细胞内脂肪酸成分检测方法 13

2.5.3 细胞外膜通透性的检测方法 14

2.5.4 纽莫康定B₀检测方法 14

2.5.5 线粒体活性的检测方法 15

2.5.6 普通分批发酵和萃取分批发酵菌丝体的形态学分析 15

第三章 结果与讨论 16

3.1 表面活性剂SDS添加对细胞内外纽莫康定B₀产量的影响 16

3.2 SDS对萃取发酵过程中细胞内脂肪酸含量和线粒体活性的影响 17

3.3 SDS对萃取发酵过程中G. lozoyensis形态的影响 18

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 纽莫康定B₀简介

1985年默克子公司对分离的菌株Glarea lozoyensis发酵液进行分析，得到一种具有潜其副作用小、耐药性小的特点符合未来低毒、安全以及广谱性新型抗生素的进一步发展方向。

图1-1 纽莫康定B₀和卡泊芬净化学结构

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