论文总字数：30412字

摘 要

 低聚果糖(Fructooligosaccharides,FOS)又称为果寡糖、果糖低聚糖或果聚糖，是一种益生元，也是一种水溶性膳食纤维。它是由蔗糖和1-3个果糖基通过β-2,1或者β-2,6糖苷键连接而成的。低聚果糖在高等植物中，特别是一些珍贵药用植物中广泛存在，但构型不同。自然存在于各种植物中的果聚糖主要是β(2→1)糖苷键连接的蔗果型(Kestose型)，β(2→6)糖苷键连接的异蔗果型(Isokestose型)，以及β(2→1)和β(2→6)糖苷键连接而成的新蔗果型(Noekestose)三种或它们的混合体。低聚果糖具有减少血氨浓度、促进双歧杆菌增殖，调整肠道菌群、增强免疫能力、增进矿物质吸收等功能。因此被广泛应用于食品、饮料、化妆品、饲料、医药等领域。

蔗果型低聚果糖的功效已广为认知，但越来越多的研究证实异蔗果型或新蔗果型低聚果糖比传统的工业生产的蔗果型低聚果糖具有更优的功效。在前期研究中本实验室已筛选出来源于Arthrobacter arilaitensis NJEM01耐有机溶剂β-呋喃果糖苷酶，用其催化蔗糖，产物仅生成了单一的6-蔗果三糖，没有蔗果四糖以及蔗果五糖生成，且转化率相对于工业生产的要求还有待进一步提高。基于此，本研究对前期通过对接等方法筛选到的起关键作用的第218位丝氨酸通过反向PCR法将其定点突变为丙氨酸，获得β-呋喃果糖苷酶的突变体菌株，用其以蔗糖为底物进行催化发现产物蔗果三糖的量相比于野生型有所提高，且催化生成了蔗果四糖，这是野生型酶原先不能催化生成的。本研究还进一步研究了β-呋喃果糖苷酶突变酶的酶学性质，突变体β-呋喃果糖苷酶的最适pH为6.5，在pH在6.0到8.5的范围内酶活也基本稳定，相对酶活力为75%左右。突变体β-呋喃果糖苷酶的最适温度为45℃左右，在温度低于35℃下处理2h后活力保留80%。相比于野生型β-呋喃果糖苷酶，突变体β-呋喃果糖苷酶的稳定性有所下降。

关键词：β-呋喃果糖苷酶 低聚果糖 定点突变

Research of the directional transformation of β - fructofuranosidase and its enzymatic properties

ABSTRACT

Fructooligosaccharides (FOS), also known as fructooligosaccharides, fructose oligosaccharides or fructans, is recognized as a prebiotics, but also a water-soluble dietary fiber. It is composed of kestose, tetrasccharide, pentasaccharides and itsmixture combined with sucrose and 1-3 fructose through β-2,1-bond or β-2,6-bond. Fructooligosaccharides are widely found in higher plants, especially in some valuable medicinal plants, but have different configurations. The fructans, which are naturally present in various plants, are predominantly β (2 → 1) glycosidic linkages of Kestose type, β (2 → 6) glycosidic linkages, Isokestose type, And Noekestose, in which β (2 → 1) and β (2 → 6) glycosidic bonds are bonded, or a mixture thereof. Fructooligosaccharides have the function of reducing blood ammonia concentration, promoting the proliferation of Bifidobacterium, adjusting intestinal flora, enhancing immunity, and enhancing mineral absorption. So it is widely used in food, beverage, cosmetics, feed, medicine and so on.

The efficacy of canola-type fructooligosaccharides has been widely recognized, but more and more studies have shown that canola fruit or fructose-fructose-type fructooligosaccharides are superior to traditional industrial production of canola type fructooligosaccharides effect. In the previous study, our laboratory has screened the anti-organic solvent β-fructofuranosidase from Arthrobacter arilaitensis NJEM01, which catalyzes the sucrose. The product only produces single 6-kestose.Fruit pentasaccharide production, and the conversion rate relative to the requirements of industrial production has yet to be further improved. Based on this, the 218 position

serine, which is a key role selected by docking and other methods, was used to mutate the mutated strain of β-fructofuranosidase by reverse-PCR. The amount of sucrose trisaccharide found in sucrose as a substrate was higher than that of wild type and catalyzed to produce sugars, which was not catalyzed by wild-type enzymes. In this study, the enzymatic properties of β-fructofuranosidase mutant enzymes were further studied. The optimum pH of the mutant β-fructofuranosidase was 6.5 and the activity was almost stable from pH 6.0 to 8.5. Relative enzyme activity is about 75%. The optimal temperature of the mutant β-fructofuranosidase was about 45 ℃, and the relative enzyme activity was kept at 80% after 2h treatment at a temperature lower than 35℃. Compared to wild-type β-fructofuranosidase, the stability of the mutant β-fructofuranosidase has decreased.

Key words: β-fructofuranosidase ; fructooligosaccharides; site-directed mutation

目 录

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

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

目录..........................................................................................................IV

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

1.1 低聚果糖概述...................................................................................1

1.1.1 低聚果糖简介...........................................................................1

1.1.2 低聚果糖生理功能....................................................................1

1.1.3 低聚果糖合成方法概述.............................................................2

1.1.4 高纯度低聚果糖的生产.............................................................2

1.2 β-呋喃果糖苷酶概述.........................................................................3

1.2.1 β-呋喃果糖苷酶的来源与特性....................................................3

1.2.2 β-呋喃果糖苷酶的结构及催化机制..............................................4

1.3 糖苷酶高合成性能改造................................................................6

1.3.1 催化位点氨基酸的定点突变......................................................6

1.3.2 催化位点以外氨基酸的定点突变................................................6

1.3.3 C端缺失突变.............................................................................6

1.4 本课题研究目的及意义.....................................................................7

1.4.1 研究意义..................................................................................7

1.4.2 研究内容..................................................................................7

1. 实验材料与方法..........................................................................8

2.1 材料与仪器.....................................................................................8

2.1.1 菌株与质粒..............................................................................8

2.1.2 酶与试剂..................................................................................8

2.1.3 主要仪器设备...........................................................................8

2.1.4 主要溶液成分与培养基.............................................................9

2.2 实验方法........................................................................................9

2.2.1 质粒提取..................................................................................9

2.2.2 β-呋喃果糖苷酶突变体引物设计.................................................9

2.2.3 β-呋喃果糖苷酶基因的定点突变...............................................10

2.2.4 转化及验证.............................................................................11

2.2.4.1 转化.................................................................................11

2.2.4.2 验证.................................................................................11

2.2.5 β-呋喃果糖苷酶突变体的诱导表达............................................11

2.2.6 蛋白SDS-PAGE分析...............................................................12

2.2.7 β-呋喃果糖苷酶突变酶酶活测定...............................................13

2.2.8 β-呋喃果糖苷酶突变酶催化合成低聚果糖的研究.......................13

2.2.9 β-呋喃果糖苷酶突变酶的酶学性质研究.....................................14

2.2.9.1 β-呋喃果糖苷酶突变酶最适反应pH....................................14

2.2.9.2 β-呋喃果糖苷酶突变酶的pH稳定性....................................14

2.2.9.3 β-呋喃果糖苷酶突变酶最适反应温度..................................14

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