论文总字数：17630字

摘 要

白藜芦醇是一种多酚类化合物，具有诸多的生物学功能。但是其较低的水溶性极大地限制了白藜芦醇作为有潜力的药物进行临床开发应用方面的生物利用度。虎杖苷是白藜芦醇3-O-β-葡萄糖苷，溶解度是白藜芦醇的31.5倍，并且抗氧化活性相当，同时功效相近。利用糖基转移酶催化白藜芦醇合成虎杖苷可以有效改善白藜芦醇水溶性低和生物利用度差的问题。实验室前期发掘的糖基转移酶UGT_BL1具有白藜芦醇高效糖基化能力，但倾向于合成白藜芦醇4’-O-葡萄糖苷，不能直接用于虎杖苷的生物合成。

本研究基于蛋白模拟和对活性口袋周围氨基酸位点的丙氨酸扫描结果，选择对区域选择性具有显著影响的两个位点进行联合突变，并对突变体蛋白的催化性质进行验证分析，最终获得的I62G N73Y位联合突变体在可溶性表达不受影响的情况下，其对白藜芦醇的3位和4’位羟基的选择性从原先的7:1提高到16:1。具体实验内容如下：

1. 对前期丙氨酸扫描突变体催化白藜芦醇糖基化的结果进行分析，找出对白藜芦醇区域选择性影响较大的位点。

2. 对使得UGTBL1区域选择性发生翻转的I62位进行饱和突变，并通过催化实验验证对应突变体区域选择性，获得了3-OH选择性进一步提高的突变体I62G。

3. 在I62G的基础上，结合73位新一轮的饱和突变，最终构建了I62G N73Y这一联合突变体蛋白，其对3-OH选择性与野生型相比提高了112倍，且可溶性表达没有收到影响。

关键词：白藜芦醇 糖基转移酶 区域选择性 蛋白质工程改造

Study on the Selectivity of Resveratrol in UGTBL1 Catalyzed by Joint Mutation

ABSTRACT

Resveratrol is a highly bioactive polyphenolic compound, but its low water solubility greatly limits the bioavailability of resveratrol as a potential drug for clinical development and application. Polydatin is resveratrol 3-O-β-glucoside, its solubility is 31.5 times that of resveratrol. They all have antioxidant activity and similar effects. So the resveratrol as a glycoside ligand synthesis of polydatin can solve this problem. The glycosyltransferase UGT_BL1, which has been explored in the laboratory, has high glycosylation ability of resveratrol, but tends to synthesize resveratrol 4'-O-glucoside and can not be directly used for biosynthesis of polydatin.

In this study, based on the protein simulation and alanine scanning of the amino acid sites around the active pockets. Two sites with significant effects on the regional selectivity were selected for the combined mutation. And the catalytic properties of the mutant protein were verified. The selectivity of the I62G N73Y-position mutant was not affected by the soluble expression of the mutant, and the selectivity of the 3 and 4 'hydroxyl groups of resveratrol was 1: 7 to 16: 1. Specific experimental content is as follows:

1. The results of glycosylation of resveratrol catalyzed by alanine scanning mutant were analyzed to find out the sites with great influence on resveratrol region selectivity.

2. The mutant I62G with 3-OH selectivity was further amplified by catalytic experiments to verify the mutant region selectivity for the I62 position where the UGTBL1 region was selectively inverted.

3. On the basis of I62G, combined with a new round of saturation mutation of 73, the combined mutant protein of I62G N73Y was constructed. The selectivity of 3-OH was 112 times higher than that of wild type, and the soluble expression was not Receive the influence.

KEYWORDS： Resveratrol ; Glycosyltransferase ; Regioselectivity； Protein engineerin

目 录

摘 要 I

ABSTRACT II

第一章 文献综述 1

1.1白藜芦醇 1

1.1.1 白藜芦醇的介绍 1

1.1.2白藜芦醇的药用价值 1

1.2虎杖苷 1

1.2.1虎杖苷简介 1

1.2.2虎杖苷的药理作用以及市场前景 1

1.3 糖基转移酶 2

1.4 糖基转移酶蛋白改造研究进展 3

1.5前期实验工作 3

1.5.1同源建模 3

1.5.2 丙氨酸扫描验证关键位点 4

第二章 糖基转移酶UGT_BL1糖基化区域的选择性改造 5

2.2 实验材料 5

2.2.1菌株、酶、缓冲液和培养基 5

2.2.2 实验试剂及实验仪器 6

2.3 实验方法 7

2.3.1 UGT_BL1 I62位饱和突变 7

2.3.2糖基转移酶突变体的表达 8

2.3.3 糖基转移酶突变体的表达检测 9

2.3.4 糖基转移酶突变体催化糖基化反应 9

2.4 结果与讨论 9

2.4.1 糖基转移酶突变体的表达与糖基化结果 9

2.4.2全质粒PCR结果分析 10

2.4.3菌落PCR结果分析 11

2.4.4糖基转移酶突变体的表达检测结果分析 12

2.4.5 I62位定点饱和突变所获UGT_BL1突变体选择性结果 13

第三章 糖基转移酶UGT_BL1的I62G N73位联合突变 15

3.1前言 15

3.2实验材料 15

3.2.1菌株、酶、缓冲液和培养基 15

3.2.2 实验试剂及实验仪器 15

3.2.3 实验方法 15

3.3结果与讨论 15

第四章 结论与展望 18

4.1 结论 18

参考文献 20

致 谢 23

第一章 文献综述

1.1白藜芦醇

1.1.1 白藜芦醇的介绍

白藜芦醇是天然产物虎杖苷的苷元，是多羟基酚类的一种化合物^[1]。其分子式为C₁₄H₁₂O₃，结构如图1.1所示。

科学家首先在葡萄中发现了它的身影，作为一种重要的抗毒素。随着人们的深入研究，陆续在花生，蓝莓，桑葚等植物中也发现了白藜芦醇的存在，也为白藜芦醇的提取开拓了更多的途径^[1]。其中当属葡萄皮中含量最高，为50～100mg/ kg。

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