论文总字数：21915字

摘 要

卤醇脱卤酶自发现以来，展现出了许多重要的用途，比如催化合成降血脂药阿托他汀侧链基团(R)-4-氰基-3-羟基丁酸乙酯、催化制备手性环氧氯丙烷和β-卤代醇。但是目前为止对卤醇脱卤酶的研究仍有许多不足，比如已经发现的具有优良催化性能的卤醇脱卤酶太少。为了解决这些问题，还需要对卤醇脱卤酶进行更加深入的研究，挖掘出更多的具有优良催化性能的新卤醇脱卤酶，并对现有催化效果良好的卤醇脱卤酶进行突变位点改造进一步提升其催化效果。

为了发现具有优良催化性能的新卤醇脱卤酶，本文首先基于数据库进行了卤醇脱卤酶的基因挖掘。以HheA、HheB、HheC的序列为模板在GenBank数据库通过基因挖掘寻找新的卤醇脱卤酶，最后挖掘出10种新酶，为了验证本次基因挖掘的效果，选择新酶HHDH10重组到E.coli BL21(DE3)中表达， 最后测出新酶HHDH10比活力为0.33 U/ml。

为预测HHDH10的三级结构以及可突变的关键残基位点，对HHDH10进行了生物信息学分析。用SWISS-MODELL对HHDH10进行同源建模，4-氯-3-羟基丁酸乙酯为配体，与其进行分子对接，选择最佳的pose。将分子对接后的模型提交至在线工具HOTSPOT WIZARD3.0进行热点分析，据热点残基的分析计算，Phe-194、Glu-197、Pro-93、Pro-94、Cys-92、Glu-109、Tyr-193的可突变预测等级为9。

关键词：卤醇脱卤酶 基因挖掘 同源建模 分子对接 热点分析

Gene mining and bioinformatics analysis of halohydrin dehalogenase

Abstract

Since the discovery of halohydrin dehalogenase, it has shown many important uses, such as the catalytic synthesis of the side chain group (R) -4-cyano-3-hydroxybutyrate ethyl ester of hypolipidemic drug atorastatin, Epichlorohydrin and β-halohydrin. However, there are still many deficiencies in the research of halohydrin dehalogenase so far. For example, there have been too few halohydrin dehalogenases with excellent catalytic performance. In order to solve these problems, it is also necessary to conduct more in-depth research on halohydrin dehalogenase, to dig out more new halohydrin dehalogenases with excellent catalytic performance, and to carry out on the existing halohydrin dehalogenases with good catalytic effect The mutation site modification further enhances its catalytic effect.

In order to discover new halohydrin dehalogenase with excellent catalytic performance, this paper firstly carried out gene mining of halohydrin dehalogenase based on the database. Using the sequence of HheA, HheB, and HheC as templates in the GenBank database to find new halohydrin dehalogenase through gene mining, and finally found 10 novel. In order to verify the effect of this gene mining, the novel HHDH10 was selected and recombined into E. It was expressed in coli BL21 (DE3), and finally the specific activity of the novel HHDH10 was 0.33 U / ml.

In order to predict the tertiary structure of HHDH10 and the positions of key residues that can be mutated, bioinformatics analysis was performed on HHDH10. HHDH10 was homologously modeled with SWISS-MODELL, ethyl 4-chloro-3-hydroxybutyrate as a ligand, and molecular docking was performed to select the best pose. Submit the model after molecular docking to the online tool HOTSPOT WIZARD3.0 for hot spot analysis. According to the analysis of hot spot residues, Phe-194, Glu-197, Pro-93, Pro-94, Cys-92, Glu-109, Tyr-193 has a mutation predictive rating of 9.

Key Words: halohydrin dehalogenase ; gene mining ; homology modeling ; molecular docking ; hot spot analysis

目 录

摘 要 Ⅰ

Abstract Ⅱ

第一章 文献综述 1

1.1卤醇脱卤酶的概述 1

1.1.1卤醇脱卤酶的简单介绍 1

1.1.2卤醇脱卤酶的结构特征 1

1.1.3卤醇脱卤酶的催化机理 2

1.2卤醇脱卤酶的基因挖掘 3

1.3生物信息学 4

1.3.1生物信息学的简介 4

1.3.2蛋白质结构预测 4

1.4本课题研究的目的和意义 5

第二章 卤醇脱卤酶的基因挖掘和生物信息学分析 6

2.1前言 6

2.2 实验材料 7

2.2.1实验试剂与酶 7

2.2.2实验仪器 8

2.2.3菌株与质粒 8

2.3 实验方法 9

2.3.1卤醇脱卤酶的基因挖掘 9

2.3.2卤醇脱卤酶的表达载体的构建 10

2.3.3卤醇脱卤酶的蛋白表达 10

2.3.4卤醇脱卤酶的酶活测定 10

2.3.5蛋白质浓度的测定 11

2.3.6分析方法 11

2.3.7卤醇脱卤酶的序列分析 12

2.3.8卤醇脱卤酶的同源建模 12

2.3.9卤醇脱卤酶的分子对接 12

2.3.10卤醇脱卤酶的热点分析 12

2.4结果与讨论 12

2.4.1卤醇脱卤酶的基因挖掘 12

2.4.2卤醇脱卤酶的表达载体的构建 13

2.4.3卤醇脱卤酶的蛋白分析 14

2.4.4卤醇脱卤酶的酶活分析 15

2.4.5卤醇脱卤酶的同源建模 15

2.4.6卤醇脱卤酶的分子对接 17

2.4.7卤醇脱卤酶的热点分析 17

第三章 主要结论与前景展望 19

3.1 主要结论 19

3.2 展望 19

参考文献 20

致谢 24

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