论文总字数：20836字

摘 要

甜菊糖苷是从甜叶菊中分离出来复合糖苷，甜度高，热量低。同时，其还具有降血糖、抗肿瘤等药理性作用，现在被广泛应用于食品和制药工业当中。目前，有40多种甜菊糖苷从甜叶菊中被提取出来。其中，甜菊苷是其主要成分，占60%-70%，甜度较高，但后苦涩味较重。莱胞迪苷E在甜叶菊中占0.2%左右，含量很少，甜度较高，且其口感相比甜菊苷更接近于蔗糖。利用传统化学工艺提取方法得到甜菊糖苷的方法产量低且成本高，无法满足莱胞迪苷E的市场供应生产需求。本研究通过采用生物酶法转化甜菊苷生成莱胞迪苷E，选取糖基转移酶UGTSL2与蔗糖合酶StSUS1进行双酶偶联，构建pRSFDuet-UGTSL2-StSUS1,导入宿主大肠杆菌进行产酶，并对催化反应体系进行优化。在pH为7.2,甜菊苷浓度为20 g/L，体系为10 mL的固定条件下，反应14个小时，研究不同物料比、温度和蛋白浓度对甜菊苷转化生成莱胞迪苷E反应的影响。结果表明，当甜菊苷与蔗糖的比例为1：5、温度为40℃、蛋白浓度为9 mg/mL 的条件下，甜菊苷能够较好的生成莱胞迪苷E。

关键词：甜菊糖苷 甜菊苷 糖基转移酶 生物转化

Biotransformation of stevioside to Rebaudioside E

Abstract

Steviol glycosides is a complex glycoside isolated from Stevia rebaudiana with high sweetness and low calories. At the same time, it also has the pharmacological effects of lowering blood sugar and anti-tumor, so it is widely used in the food and pharmaceutical industries. Currently, more than 40 different sweetness steviol glycosides have been extracted from Stevia rebaudiana. Among them, the main component of steviol glycosides is stevioside,accounting for 60-70%, its sweet taste is pure, the taste is closer to sucrose,but the bitter taste is heavier. Rebaudioside E is rare in stevia,but has a higher sweetness,and its taste is better than stevioside and there is no bitter taste. The glycosyltransferase UGTSL2 was ligated with the sucrose synthase StSUS1 to construct the double-enzyme expression plasmid pRSFDuet-UGTSL2-StSUS1,which was introduced into host E. coli for enzyme production. The glycosyltransferase UGTSL2 was used to catayze the synthesis of stevioside.The performance of reaction system for the biosynthesis of Rebudioside E,and the effect of rection conditions on the yield of the product were determined. The pH was 7.2, the concentration of stevioside was 20 g/L,and the system was reacted for 14 hours under fixed conditions of 10mL. The effects of different material ratios, temperature and protein concentration on the reaction of stevioside to sucrose was 1:5, the temperature was 40 ℃, and the protein concentration was 9mg/mL, stevioside was able to generate Rebaudioside E.

Key Words: steviol glycoides; stevioside; glycosyltransferase; biotransformation

目 录

摘要 II

Abstract III

第一章 文献综述 1

1.1 前言 1

1.2 甜菊苷及莱胞迪苷E的获取方法 1

1.3 糖基转移酶合成糖苷类化合物 2

1.4 蔗糖合酶和糖基转移酶偶联 2

1.5 本论文的立题意义和研究内容 3

第二章 双酶偶联系统的构建、表达及反应体系的建立 4

2.1 前言 4

2.2 实验材料 5

2.2.1 菌株与质粒 5

2.2.2 工具酶，分子量标准和试剂盒 5

2.2.3 实验试剂 5

2.2.4 实验仪器 6

2.3 试验方法 7

2.3.1 培养基 7

2.3.2 菌株培养 7

2.3.3 粗酶液的获取 7

2.3.4 糖基转移酶的SDS-PAGE 分析 8

2.3.5 糖基转移酶活性测定 8

2.3.6 蔗糖合成酶活性测定 8

2.3.7 双酶催化反应 8

2.3.8 甜菊苷、莱胞迪苷E标品溶液的配置 8

2.3.9 甜菊苷及莱胞迪苷E HPLC测定 8

2.3.10 甜菊苷转化生成莱胞迪苷E分析 9

第三章 实验结果与讨论 10

3.1 双基因表达质粒验证 10

3.2 甜菊苷和莱胞迪苷标准曲线 10

3.3 甜菊苷和莱胞迪苷标准曲线 11

3.4 甜菊苷转化合成莱胞迪苷E条件优化………………………………………………12

3.4.1 不同物料比 ……………………………………………………………………12

3.4.2 不同温度……………………………………………………………………… 13

3.4.3 不同蛋白浓度………………………………………………………………… 13

第四章 实验不足与展望 16

4.1 实验结论 16

4.2 实验不足与展望 16

参考文献 17

致谢 20

第一章 文献综述

1.1 前言

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