论文总字数：20579字

摘 要

几丁质酶（Chitinase，EC 3.2.1.14）广泛存在于各种生物中，包括细菌、真菌、昆虫和植物等生物。它是一种通过水解其底物几丁质（Chitin）的β-1，4-糖苷键产生几丁质寡糖和N-乙酰葡萄糖胺的糖苷水解酶。几丁质又称甲壳素，是由N-乙酰-β-D-葡萄糖胺通过β-1,4糖苷键连接构成的直链同多糖。几丁质广泛存在于生物界，是自然界中第二丰富的多糖，仅次于纤维素。几丁质的中间降解产物几丁寡糖（CHOS）具有高度的生物相容性和无毒性，具有抗病毒、抗菌、抗真菌、免疫调节和抗氧化活性。因此，CHOS在食品加工、生物医学、农业、净水和化妆品等各个领域都有着广阔的应用前景。

然而，由于野生型菌株中的几丁质酶产量低和活性弱，这限制了其催化降解几丁质的能力，几丁质酶的实际应用仍然具有挑战性。

本文从实验室保藏的深海微生物中筛选出可降解几丁质的微生物，得到高活性的几丁质酶基因序列CTS1，进行在线翻译生成了几丁质酶序列CTAS1，结合分子模拟和蛋白质工程技术构建了目标几丁质酶CT1的三维结构模型，通过分子对接，预测其催化区与结合区，分析得到关键位点残基为催化残基附近Trp₁₁₄（4.5Å以内）以及结合口袋的袋口残基Trp₂₉₀。最后通过模拟改变关键位点的残基，获得了模拟突变体MCT2、MCT3、MCT4和MCT5，通过计算机模拟技术同源建模，分析其结合区与催化区，发现MCT2(Trp₁₁₄→Thr₁₁₄)和MCT3（Trp₂₉₀→Gly₂₉₀）模拟突变体的结合结构域都被破坏，而 MCT4 (Trp₁₁₄→Tyr₁₁₄)和MCT5（Trp₂₉₀→Phe₂₉₀）的突变体的催化区和结合结构域都发生了不同程度的改变，极大可能提高了的几丁质酶CT1降解结晶几丁质的能力。

关键词：几丁质酶；定向进化；同源建模；理性设计

Rational design scheme of chitinase directed evolution

Abstract

Chitinase (Chitinase, EC 3.2.1.14) is widely present in various organisms, including bacteria, fungi, insects and plants. It is a glycoside hydrolase that produces chitin oligosaccharides and N-acetylglucosamine by hydrolyzing the β-1,4-glycosidic bond of its substrate chitin. Chitin is a linear homopolysaccharide composed of N-acetyl-β-D-glucosamine linked by β-1,4 glycosidic bonds. Chitin exists widely in the biological world and is the second most abundant polysaccharide in nature, second only to cellulose. Chitin oligosaccharide (CHOS), an intermediate degradation product of chitin, is highly biocompatible and non-toxic, and has antiviral, antibacterial, antifungal, immunomodulatory and antioxidant activities. Therefore, CHOS has broad application prospects in various fields such as food processing, biomedicine, agriculture, water purification and cosmetics.

However, due to the low yield and weak activity of chitinase in wild-type strains, which limits its ability to catalyze the degradation of chitin, the practical application of chitinase remains challenging.

In this study, microbes degrading chitin were selected from the deep-sea microorganisms deposited in the laboratory, and the highly active chitinase gene sequence CTS1 was obtained. The chitinase sequence CTAS1 was generated by online translation, combining molecular simulation and protein engineering The three-dimensional structure model of the target chitinase CT1 was constructed by technology, and the catalytic region and the binding region were predicted by molecular docking. The key site residues were Trp114 (less than 4.5Å) near the catalytic residue and the pocket of the binding pocket. Finally, by changing the residues at key positions, simulated mutants MCT2, MCT3, MCT4 and MCT5 were obtained. The homologous modeling of the computer simulation technology was used to analyze the binding region and the catalytic region. (Trp₂₉₀→Gly₂₉₀) The binding domains of the simulated mutants were destroyed, while the catalytic regions and binding domains of the mutants of MCT4 (Trp₁₁₄→Tyr₁₁₄₎ and MCT5 (Trp₂₉₀→Phe₂₉₀) were changed to different degrees Possibly increased the ability of chitinase CT1 to degrade crystalline chitin.

Keywords: chitinase; directed evolution; homology modeling; rational design

目录

摘要 I

Abstract II

目录 I

第一章 前言 1

第二章 文献综述 2

2.1 几丁质酶概述 2

2.1.1 几丁质酶的基本介绍 2

2.1.2 几丁质酶的分类 2

2.1.3几丁质酶的氨基酸序列特征 3

2.1.4 18家族几丁质酶的三维结构 3

2.1.5 几丁质酶的催化机制 3

2.2蛋白质工程概述 4

2.2.1定向进化 5

2.2.2蛋白质理性设计 5

2.2.3蛋白质半理性设计 5

2.4课题来源及研究内容 6

2.4.1课题来源 6

2.4.2本课题研究内容 6

第三章 材料与方法 8

3.1几丁质酶基因 8

3.2几丁质酶氨基酸残基分析与信号肽预测 8

3.3多序列比对与进化树分析 9

3.4同源建模与模型评估 9

3.5分子对接 9

第四章 结果与讨论 10

4.1几丁质酶CT1的氨基酸残基组成分析与信号肽预测 10

4.2系统发生树构建与多序列比对分析 10

4.3同源建模与分子对接 13

4.4关键位点残基改变与影响分析 16

4.5讨论与展望 21

参考文献 23

致谢 26

第一章 前言

几丁质（Chitin）是由β-1,4糖苷键连接的N-乙酰葡萄糖胺(GlcNAc)单体组成的同多糖，是自然界中第二普遍存在的多糖，仅次于纤维素。它是广泛生物的主要结构组成部分，其中几丁质含量最丰富的是真菌和节肢动物。据报道，世界各地的海产业每年产生约六至八百万吨壳废料^[1]。几丁质数量大、生化特性显著，但几丁质的商业应用范围有限，这主要是由于几丁质的高结晶度和不溶性使加工变得困难^[2]。另外，几丁质的降解是一项耗时而艰巨的任务，处理含有几丁质的废物已然成为一项环境挑战，并即将成为许多国家的危机。

因此，必须找到一种有效和环保的方法来降解几丁质废物，并且最好将其价值化。几丁寡糖（lt;20）是一种低聚合度的几丁质衍生物，由于其溶解性的提高，具有很大的价值。此外，它们具有抗病毒、抗菌、抗真菌、免疫调节和抗氧化活性。所以，在食品加工、生物医学和生物防治等各个领域都有着广阔的应用前景^[3]。

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