论文总字数：26612字

摘 要

酪醇是红景天苷糖基化反应的苷元底物分子，其中的红景天苷已经被广泛的应用于食品、医药等重要领域。酪醇的糖基化产物的分离制备还有很大开发空间，如何高效经济的获得糖基化产物一直是热门研究方向。本课题利用糖基转移酶建立全细胞转化体系，来制备获得酪醇的糖基化产物，并对其进行分离条件的优化。主要研究：

解决了活化酶UPD-葡萄糖来源的问题。并且成功构建了糖基转移酶UGT_BL1的全细胞表达体系。

考察糖基供体前体葡萄糖和底物受体酪醇浓度对全细胞转化的影响。除此之外，还尝试了工程菌全细胞重复批次使用的可能性，为全细胞转化工艺中细胞的固定化及重复利用做了前期探索。

对糖基化产物进行结构鉴定与分析，利用放大的全细胞转化体系合成酪醇糖苷产物，并经过萃取分离和制备色谱分离以及进一步的结构鉴定。最终确定糖基化产物为红景天苷及Icariside D2。

关键词：酪醇 糖基转移酶 红景天苷 萃取分离 制备色谱

ABSTRACT

Tyrosol is the glycosylation of glycosides in the glycosylation substrate, which has been widely used in food, medicine and other important areas.The separation of the glycosylated product of tyrosine also has a great deal of development space, and how efficient and economical access to glycosylation products has been a hot research direction.In this study, a whole cell transformation system was established using glycosyltransferase to prepare the glycosylated product of tyrosol, and the separation conditions were optimized.The main research is as follows:

Solve the problem of activated enzyme UDP-glucose source.And the whole cell expression system of glycosyltransferase UGT_BL1 was successfully constructed.

To investigate the effect of glycosyl concentration of glucose and substrate receptor on whole cell transformation. In addition, the possibility of repeating the use of whole cells in engineering bacteria was also attempted, and the immobilization and reuse of cells in the whole cell transformation process were explored.

The glycosylation products were identified and analyzed, and the product was synthesized and purified by extraction and preparative chromatographic separation and further structural identification.The final determination of glycosylation products for salidroside and icariside D2.

Key Words: Tyrosol；Glycosyltransferase；Salidroside；Extraction separation；Preparation of chromatography

目 录

摘要.............................................................................................................I

ABSTRACT...............................................................................................II

第一章 文献综述......................................................................................1

1.1糖基转移酶概述..................................................................................................1

1.1.1 糖基转移酶的挖掘方法进展....................................................................2

1.1.2 糖基转移酶的改造研究进展....................................................................2

1.2 红景天苷的医药用途.........................................................................................3

1.3 本课题研究的内容及意义.................................................................................4

1.3.1 本课题研究的内容....................................................................................4

1.3.2 本课题研究的意义....................................................................................4

第二章 糖基转移酶工程菌构建..............................................................5

2.1 前言.....................................................................................................................5

2.2 实验材料.............................................................................................................5

2.2.1 实验试剂....................................................................................................5

2.2.2 糖基转移酶及菌株....................................................................................5

2.2.3 构建糖基转移酶UGT_BL1工程菌.............................................................5

2.3 实验方法.............................................................................................................6

2.3.1 构建糖基转移酶UGT_BL1工程菌............................................................6

2.3.2 糖基转移酶UGT_BL1工程菌全细胞的制备............................................6

2.4 结果与讨论.........................................................................................................6

2.5 本章小结.............................................................................................................7

第三章 全细胞催化酪醇糖基化反应.......................................................8

3.1 前言.....................................................................................................................8

3.2 实验材料.............................................................................................................8

3.2.1 实验试剂....................................................................................................8

3.2.2 实验仪器....................................................................................................8

3.3 实验方法.............................................................................................................8

3.3.1 全细胞催化体系的建立............................................................................8

3.3.2 工程菌全细胞重复批次使用....................................................................9

3.3.3 样品的取样与检测..................................................................................9

3.3.4 工程菌胞内底物及产物的检测..............................................................9

3.4 结果与讨论.........................................................................................................9

3.4.1 葡萄糖浓度对全细胞转化酪醇的影响....................................................9

3.4.2 底物浓度对全细胞转化酪醇的影响......................................................10 3.4.3 糖基转移酶工程菌全细胞重复批次使用.............................................11

3.5 本章小结 ..........................................................................................................12

第四章 产物分离与鉴定.....................................................................................14

4.1 前言...................................................................................................................14

4.2 实验材料...........................................................................................................14

4.2.1 实验仪器..................................................................................................14

4.3 实验方法 ..........................................................................................................14

4.3.1 全细胞催化产物的分离纯化...................................................................14

4.3.2 产物结构鉴定...........................................................................................15

4.4 结果与讨论.......................................................................................................15

4.4.1 糖基化产物胞外分布..............................................................................15

4.4.2 全细胞转化法制备红景天苷与其它方式的比较..................................16

4.4.3 糖基化产物的质谱分析和核磁共振谱分析..........................................17

4.5 本章小结...........................................................................................................23

第五章 结论与展望..............................................................................................24

5.1 结论...................................................................................................................24

5.2 展望...................................................................................................................24

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