论文总字数：15757字

摘 要

本课题对两株产紫杉醇的好氧型内生真菌MF5和MF8进行种子液培养和发酵培养，并采用GA、MeJA、CuSO₄三种诱导因子处理发酵液。发酵条件为：发酵罐温度设置为恒定28℃；转速调节为120 rpm/min；pH为7.00；溶氧值设为100%。在发酵培养阶段，观察分析了真菌微观形态，测定pH和沉降比变化；利用HPLC法检测真菌发酵产紫杉醇含量。初步建立了内生真菌MF5和MF8液体发酵制备紫杉醇的高产培养方式。

结果表明：添加诱导剂能明显增加发酵液中紫杉醇产量。（1）MF5和MF8单独添加MeJA、GA、CuSO₄后发酵液中的紫杉醇含量均有提升：其中添加MeJA的效果最佳，MF5和MF8发酵获得的紫杉醇分别达到0.832 mg/L和1.794 mg/L；添加CuSO₄的效果最差，产量分别为0.434 mg/L和1.437 mg/L。而不加诱导剂处理下，菌株MF5 和MF8的紫杉醇产量分别仅有0.318 mg/L和1.212 mg/L；（2）菌株MF5和 MF8均在三种诱导剂全部添加、发酵培养60 h时紫杉醇产量最高。其中MF5达2.719 mg/L，MF8达 2.537 mg/L。

关键词：紫杉醇 内生真菌 诱导剂 HPLC检测

Taxus Endogenous Fungi Producing Taxol

Multiple induction and yield correlation analysis

Abstract

In this dissertation, two kinds of aerobic endophytic fungi MF5 and MF8 producing paclitaxel were cultured in seed fluid and fermentor; and the fermentation media was treated with GA, MeJA and CuSO₄. Parameters were as follows: the fermentor temperature was set to be constant at 28°C; Rotating speed was 120 rpm/min; pH value was 7.00 and DO value was set to 100%. During the fermentation period, the microscopic morphology was observed; Besides, pH change and settling ratio were mearsured at the same time. The production of taxol was calculated by HPLC detecting method. Then, the high-yield culture method for the production of taxol by liquid fermentation of endophytic fungi MF5 and MF8 was preliminarily established.

The results reveal that the addition of inducer can significantly boom the production of taxol in the fermentation media. (1) The taxol amount produced by strains MF5 and MF8 was increased after MeJA, GA and CuSO₄ were added in the fermentation media separately: Adding MeJA resulted in the highest yield, and the taxol yields of 0.832 mg/L and 1.794 mg/L was obtained by the fermentation of MF5 and MF8 respectively; The worst effect was obtained with yields of 0.434 mg/L (MF5) and 1.437 mg/L (MF8) by adding CuSO₄, respectively. However, without treatment with inducer, the taxol yields of strains MF5 and MF8 were only 0.318 mg/L and 1.212 mg/L, respectively; (2) Both strains MF5 and MF8 produced the largest amount of taxol when they were fermented for 60 h, and all the three inducers were added. Among them, the yield of taxol was up to 2.719 mg/L and 2.537 mg/L respectively when MF5 and MF8 was cultured for 60 h.

KEYWORDS: Taxol; Endophytic fungi; Inducer; HPLC detection

目 录

摘 要 I

Abstract II

第一章 文献综述 1

1.1 紫杉醇简介 1

1.1.1紫杉醇的发现 1

1.1.2紫杉醇的的结构和理化性质 1

1.2紫杉醇的生产方法 2

1.3 液体发酵提高紫杉醇产量的途径 3

1.3.1 添加诱导子 3

1.3.2 添加代谢旁路抑制剂 4

1.4 本课题研究内容、目的和意义 5

第二章 实验材料和方法 6

2.1 实验材料 6

2.1.1 菌株 6

2.1.2 培养基成分 6

2.1.3 主要药品和试剂 6

2.2 实验方法 8

2.2.1 种子培养 8

2.2.2 发酵实验 8

2.2.3 发酵罐生产工艺 8

2.2.4 发酵液培养性状分析 9

2.2.5 紫杉醇提取方法 10

2.2.6 紫杉醇含量的测定方法 10

第三章 结果与讨论 11

3.1 标准曲线的绘制 11

3.2 发酵液培养性状分析 11

3.3 诱导剂对紫杉醇产量的影响 14

3.3.1 发酵时间对紫杉醇产量的影响 16

3.3.2 不同诱导剂对紫杉醇产量的影响 18

3.4 结论与展望 20

3.4.1 结论 20

3.4.2 展望 20

参考文献 22

致谢 25

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