论文总字数：21904字

摘 要

金属有机骨架固定化酶具有比表面积大、催化活力高、稳定性高、传质阻力低等优点，但仍存在缺点，如制备过程复杂，制备体系的苛刻环境对酶构象的伤害大，催化应用范围受到限制等。本文采用生物相容性好、绿色友好的新型溶剂，即深度共熔溶剂（DES）代替传统的有机溶剂作为固定化媒介，脂肪酶与金属有机骨架经共沉淀后，简单快速地合成金属有机骨架-脂肪酶纳米复合物（CRL-Hmin@MOF），然后进行形貌和光谱分析，并对游离脂肪酶（CRL）和固定化脂肪酶（CRL-Hmin@MOF）的酶学性质进行比较研究。主要结论如下：

（1）采用共沉淀的方法，简单快速地合成了一系列CRL-Hmin@MOF，并进行表征和酶学性质的研究。根据其水解三乙酸甘油酯的活力，从DES氢键供体、DES含水量、金属离子、配体比例、酶的添加量五个方面进行固定化条件的优化，得到在氯化胆碱-甘油中合成的含有钴元素的固定化脂肪酶（CRL-Hmin@MOF）的相对酶活最高，是游离酶的1.07倍。

（2）表征分析了CRL-Hmin@MOF的形貌特征和化学组成，扫面电镜结果证明CRL-Hmin@MOF的结构是纳米形态，酶的参与使得载体金属有机骨架（Hmin@MOF）的形貌结构发生改变，光谱分析证明CRL已被成功包埋在Hmin@MOF内部。

（3）对比分析研究了游离CRL和CRL-Hmin@MOF的酶学性质。相较于游离CRL，CRL-Hmin@MOF具有更宽的温度和pH范围，且温度、pH和有机耐受性均显著增强，CRL-Hmin@MOF重复使用4次后，仍保留了50%以上的初始活力。

关键词：金属有机骨架 深度共熔溶剂 脂肪酶 固定化 酶学性质

Metal organic framework immobilized lipase in deep eutectic solvent System

ABSTRACT

Metal-organic framework immobilized enzymes have the advantages of large specific surface area, high catalytic activity, high stability, low mass transfer resistance and so on. However, there are still shortcomings, such as the complex preparation process, the harsh environment of the preparation system has a great harm to the conformation of the enzyme, and the application scope of the catalyst is limited. In this paper, a new solvent with good biocompatibility and green friendliness, deep eutectic solvent (DES), is used as an immobilization medium instead of traditional organic solvents. After the co-precipitation of lipase and metal-organic framework, the metal-organic framework-lipase(CRL-Hmin@MOF) nanocomposite is synthesized quickly and simply. The morphology and spectral analysis were then performed, and the enzymatic properties of free lipase (CRL) and immobilized lipase (CRL-Hmin@MOF) were compared. The main conclusions are as follows:

(1) Using a co-precipitation method, a series of CRL-Hmin@MOFs were synthesized in a simple and rapid manner, characterization and enzymatic properties were studied. According to the activity of the triacetin hydrolysis, the optimization of the immobilization conditions was performed from the following five aspects: DES hydrogen bond donor, DES water content, metal ion, ligand ratio, and enzyme dosage. The highest relative activity of the immobilized lipase (CRL-Hmin@MOF) containing a cobalt element synthesized in choline chloride-glycerol was obtained. And the immobilized enzyme activity was 1.07 times that of the free enzyme.

(2) Through surface features analysis the morphology and chemical composition of CRL-Hmin@MOF. Scanning electron microscopy results demonstrate that the structure of CRL-Hmin@MOF is Nanomorphic. The enzyme's participation changes the morphology of the carrier metal organic framework (Hmin@MOF). Spectral analysis demonstrated that the CRL has been successfully embedded inside Hmin@MOF.

(3) Comparative analysis of the enzymatic properties of free CRL and CRL-Hmin@MOF. Compared to free CRL, CRL-Hmin@MOF has a wider temperature and pH range, and the temperature. Besides, pH and organic tolerance are significantly enhanced. After repeated use of CRL-Hmin@MOF for 4 times, more than 50% of initial vigor is still retained.

Keywords: Metal organic framework; Deep eutectic solvent; Lipase; Immobilization; Enzymatic properties

目 录

摘要 I

ABSTRACT II

第一章 文献综述 1

1.1 脂肪酶概述 1

1.1.1 脂肪酶的简介 1

1.1.2 脂肪酶的特性 1

1.1.3 脂肪酶的应用与发展 1

1.2 酶的固定化概述 1

1.3 金属有机骨架概述 2

1.4 金属纳米材料固定化酶 2

1.4.1 纳米技术固定化 2

1.4.2 金属纳米粒子固定化酶 3

1.5 深度共熔溶剂（DES）概述 3

1.5.1 DES简介 3

1.5.2 DES应用与发展前景 4

第二章 实验材料和方法 5

2.1 实验材料和设备 5

2.2 试验方法及步骤 6

2.2.1 酶蛋白固定量的测定 6

2.2.2 酶活力测定 7

2.2.3 pH缓冲溶液的配置 8

2.2.4 金属离子的筛选 9

2.2.5 氢键受体的筛选 10

2.2.6 纳米元件的影响 10

2.2.7 固定化条件的优化 11

2.2.8 表征分析 11

2.2.9 酶学性质的研究比较 12

第三章 实验结果与讨论 14

3.1 引言 14

3.2 实验结果与分析 14

3.2.1 固载量的测定 14

3.2.2 金属离子的筛选 15

3.2.3 氢键受体的筛选 15

3.2.4 纳米元件对酶活的影响 16

3.2.5 配体比例的优化 17

3.2.6 酶添加量的优化 18

3.2.7 DES体系中含水量的优化 18

3.2.8 形貌分析 19

3.2.9 紫外可见光谱分析 20

3.2.10红外光谱分析 20

3.2.11最适反应温度 21

3.2.12最适反应pH 22

3.2.13温度稳定性 22

3.2.14 pH稳定性 23

3.2.15有机耐受性 24

3.2.16重复利用率 24

第四章 结论与展望 26

4.1 结论 26

4.2 展望 26

4.2.1 修饰改进 26

4.2.2 实际应用 27

参考文献 28

致谢 30

第一章 文献综述

1.1 脂肪酶概述

1.1.1 脂肪酶的简介

脂肪酶（Ec.3.1.1.3）作为一类可以分解大分子中酯键的水解酶，在现代生化工业被广泛应用。其作用原理也十分简单：将甘油三酯逐步催化最终分解成脂肪酸与甘油。脂肪酶通常情况下仅仅是由多肽链构成的，所以它的基本组成单位是氨基酸。显而易见：其催化活性由它们的蛋白质结构决定的^[1]。

1.1.2 脂肪酶的特性

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