论文总字数：22370字

摘 要

莱鲍迪甙D是一种来源于甜叶菊叶子中的甜菊糖甙，是一种纯天然的生物甜味剂。它甜度高，同时，还具有卡路里低的特性。由于莱鲍迪甙D在甜叶菊叶子中的含量仅能积累至不到干叶重的百分之一，因此直接由甜叶菊叶子中提取莱鲍迪甙D的方式，不仅产量低、产品纯度不高，而且生产工艺复杂繁琐致使生产成本高，经济效益差。本课题以大肠杆菌为宿主构建工程菌，令其表达糖基转移酶用以催化底物莱鲍迪甙A合成本次研究的目标产物莱鲍迪甙D。

本实验首先通过序列对比及文献阅读筛选出3条糖基转移酶序列：UGT-d1、EUGT 和UGT-SL2，分别构建表达上述三个酶的编码基因的重组质粒到pET22b-d1、pET28a-EUGT和pET28a-SL2，将构建好质粒导入到宿主细胞大肠杆菌中。采用乳糖诱导重组蛋白表达，重组菌株在30 ℃培养2 h后转25 ℃，过夜培养后收集菌体，经超声破碎离心获取上清作为粗酶液。利用Brandfrod法测出粗酶夜总蛋白浓度后，设计转化体系时的酶浓度，约为3 mg/mL。采用SDS-PAGE的方法检测到UGT-d1、EUGT 和UGT-SL2三条外源基因均在宿主中过量表达。将含有三条不同基因的重组菌分别进行单独发酵后，产物利用高效液相色谱仪进行检测。经检测结果分析发现重组酶EUGT几乎无法催化底物莱鲍迪甙A合成目标产物莱鲍迪甙D；重组酶UGT-d1催化效率较低，即使在最优条件下产量也仅有0.081 g/L;而重组酶UGT-SL2转化效率较高，反应液中莱鲍迪甙D浓度可以达到0.69 g/L。

关键词： 甜菊糖甙 莱苞迪甙D 糖基转移酶 大肠杆菌

The Study on Enzymatic Synthesis of Rebaudioside D

Abstract

Rebaudioside D is one of natural low-calories and high-potency sweeteners which belongs to steviol glycosides. Since the content of Rebaudioside D in the leaves of the stevia just can accumulate to less than one percent of dry leaf weight, the production is limited by the way of directly extracting Rebaudioside D from the stevia leaves. Furthermore, some disadvantages such as the impurity extractive which contains many substance and the tedious process also restricts its development.In this study, Escherichia coli was used as the host to construct the recombinant which expresses the glycosyltransferase that can catalyze the substrat rebaudioside A to the target rebaudioside D in this study.

In this study, three glycosyltransferase sequences: UGT-d1, EUGT and UGT-SL2 were screened by sequence comparison and literature reading. The genes: UGT-d1,EUGT and UGT-SL2UGT-d1 were correspondingly constructed into the plasmids:pET22b-SL2, pET28a-EUGT and pET28a-d1.Then these three plasmids were imported into the host: Escherichia coli to expressed the offspring. Adopting lactose to induce express protein, the crude enzyme fluid was obtained by ultrasonicating and centrifugating the recombinant strain which has been culture overnight at 25℃ after had been inducible expression at 30℃ for two hours. Using the Brandfrod method, the concentration of enzyme was about 3 mg / mL. Three foreign genes were efficiently expressed in the host according to the analysis by the method of SDS-PAGE.

The recombinant strains containing three different genes were fermented separately. The products were detected by high performance liquid chromatography (HPLC). It was found that the recombinant enzyme EUGT could hardly catalyze rebaudioside A into rebaudioside D. At the same time gene UGT-d1 had lower efficiency and the yield was only about 0.081 g / L while UGT-SL2 has higher catalytic efficiency and its yield can reach 0.69 g / L.

Key Words: Stevioside; Rebaudioside D; Glycosyltransferase; Escherichia coli

目 录

摘 要 I

Abstract II

目 录 III

第一章 文献综述 1

1.1 莱鲍迪甙A概况 1

1.2 莱鲍迪甙D性质 1

1.2.1 莱鲍迪甙D简介 1

1.2.2 莱鲍迪甙D现有生产工艺 2

1.2.3莱鲍迪甙D应用 2

1.3 糖基转移酶概况 3

1.4 莱鲍迪甙D检测方法 3

1.4.1 样品处理方法 3

1.4.2 Brandford法 3

1.4.3 SDS-PAGE法 4

1.4.4 高效液相色谱法（HPLC） 4

1.4.5 质谱检测法（MS） 5

1.5 课题意义 5

第二章 实验方法与材料 6

2.1 实验材料与实验仪器 6

2.1.1 实验仪器 6

2.1.2 实验试剂 6

2.1.3 菌株、质粒、工具酶、分子量标准和试剂盒 7

2.1.4 实验试剂配方 8

2.2 实验方法 11

2.2.1 重组质粒的的提取（pET22b-SL2、pET28a-EUGT、pET28a-d1） 11

2.2.2 双酶切验证 12

2.2.3 琼脂糖凝胶电泳 13

2.2.4 琼脂糖DNA凝胶回收 13

2.2.5 大肠杆菌BL21(DE3)感受态细胞的制备 13

2.2.6 载体和片段的连接 14

2.2.7 将重载质粒转化到大肠杆菌中 14

2.2.8 目的基因在大肠杆菌中的表达 14

2.2.9 粗酶液的获取 15

2.2.10 Brandford法测定蛋白浓度 15

2.2.11 蛋白凝胶电泳SDS-PAGE测定蛋白浓度 15

2.2.12 以粗酶液催化莱鲍迪甙A生成莱鲍迪甙D 16

2.2.13 质谱法确定产物是否为莱鲍迪甙D 16

2.2.14 高效液相色谱法测定产物莱鲍迪甙D的产量 16

第三章 结果与讨论 17

3.1 UGT-d1、EUGT 和UGT-SL2基因的双酶切验证 17

3.2 SDS-PAGE蛋白凝胶图谱 18

3.2.1 大肠杆菌BL21(DE3)中表达UGT-SL2的蛋白凝胶结果图 18

3.2.2 大肠杆菌BL21(DE3)中表达UGT-d1的蛋白凝胶结果图 18

3.2.3 大肠杆菌BL21(DE3)中表达EUGT的蛋白凝胶结果图 19

3.3 Brandford法测定酶浓度 19

3.4 产物的高效液相色谱（HPLC）鉴定 20

3.5 产物的质谱（MS）鉴定 21

第四章 结论与展望 26

4.1 结论 26

4.2 展望 26

参考文献 28

致 谢 31

第一章 文献综述

1.1 莱鲍迪甙A概况

从立体化学分子结构来看，莱鲍迪甙A在C₁₉上连接一个葡糖基和C₁₃上连接了多个不同数量的葡糖基以及鼠李糖基，从而形成了莱鲍迪甙A与众不同的风味以及高甜度的特性^[1]。莱鲍迪甙A的分子量为967，熔点为242~244 ℃。莱鲍迪甙A在酸碱性溶液中较稳定，且不仅在日光照射下也十分稳定，其在烘烤（温度约为390℃下）中的使用也表现出良好的热稳定性。莱鲍迪甙A可溶于一些有机溶剂，例如苯、醚和氯仿等。而且在水溶性上，莱鲍迪甙A相比于其它甙类，在水中具有较大的溶解性。

图1-1 莱鲍迪甙A的分子结构式

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