论文总字数：20579字

摘 要

脂肪酶是工业应用中的常用酶，在有机溶剂中能催化包括酯交换、酯化和酰胺化在内的一系列反应，并具有区域选择性、立体选择性和较高的稳定性。而脂肪酶作用的底物往往在有机相中才有较好的溶解度，但是蛋白质在有机溶剂中会变性，因此大多数酶类在有机溶剂中活力受到限制甚至失活。实现脂肪酶在有机环境下保持有较高的活性和稳定性成为了生产运用中亟待解决的难题。

本文拟通过在含有有机溶剂的脂肪酶催化反应体系中外源添加海藻糖的方式来提高脂肪酶的催化稳定性。本研究以褶皱假丝酵母脂肪酶(CRL)为脂肪酶代表，以N,N-二甲基甲酰胺(DMF)为有机溶剂代表，通过模型反应橄榄油的水解，考察了不同催化条件下脂肪酶的水解活力，并结合荧光光谱这一现代分析化学表征技术，分析海藻糖在有机环境中保护脂肪酶的热力学行为。实验结果表明，随着有机溶剂浓度的增高，脂肪酶的水解活力明显降低，荧光强度明显增强，这说明有机溶剂能够降低脂肪酶的催化稳定性。而海藻糖能够有效抑制脂肪酶在有机溶剂中变性，并呈浓度依赖行为，即随着海藻糖浓度增大，其保护脂肪酶的作用增强。

本发明涉及的海藻糖具备天然无害和化学性质稳定这两大优势。本发明提供的方法简单有效，不危害环境，且可以低成本、高效率地解决脂肪酶在工业应用中有机溶剂耐受性差的问题。

关键词：脂肪酶 海藻糖 有机溶剂耐受性 热力学分析

The study of improving lipase’s tolerance in organic solvent by exogenously adding trehalose

ABSTRACT

Lipase is an enzyme commonly used in industrial production, it can catalyze some reactions including transesterification, esterification, amidation and so on when in organic solvents environment. Lipase is characterized with regioselectivity, stereoselectivity and high stability. Substrate lipase catalyzing tend to have good solubility only in organic phase, while the protein will denature in such environment. So most of enzyme’s vitality will be restrained even inactivated in organic solvents. How to improve the stability and activity of lipase in organic solvents is the key in industrial application.

This paper plans to improve catalyst stability of lipase through exogenously adding trehalose in lipase-catalyzing reaction system containing an organic solvent. The research chooses Candida rugosa lipase(CRL) as the representative of lipases and N,N-Dimethylformamide(DMF) as the representative of organic solvents, simulates the hydrolysis reaction of olive oil in which we examines the activity of lipase in different catalytic hydrolysis conditions and analyzes thermodynamics behavior of trehalose that happens to protect lipase in organic solvents with fluorescence spectrum technology.The experimental results show that hydrolysis activity of lipase decreased and fluorescence intensity enhanced obviously with higher concentration of organic solvent, which shows that the catalytic stability of the lipase could be reduced by organic solvent.Besides, trehalose can effectively restrain denaturation of lipase in organic solvents which is related to concentration, that is, the protection effect to lipase increased as the trehalose concentration increases.

Trehalose used in this invention is non-toxic and has stable chemical properties. The method we adopt is simple ,effective, environmentally friendly and can solve the problem that lipase shows low organic solvent tolerance in industrial application with low cost and high efficiency.

KEYWORDS: lipase trehalose organic solvent tolerance thermodynamic analysis

目 录

摘 要 I

ABSTRACT II

第一章 文献综述 1

1.1 脂肪酶 1

1.1.1 脂肪酶简介 1

1.1.2 常见脂肪酶的来源 1

1.1.3 脂肪酶的应用 2

1.1.4 脂肪酶应用存在的问题 3

1.2 脂肪酶耐有机溶剂性的研究现状 3

1.2.1 固定化技术 3

1.2.2 定向进化技术 3

1.2.3 化学修饰 4

1.2.4 添加渗透剂 4

1.3 海藻糖 4

1.3.1 海藻糖的性质 4

1.3.2 海藻糖提高蛋白质稳定性的机制 5

1.4 本论文研究的目的意义和主要内容 5

1.4.1 本论文研究的目的和意义 5

1.4.2 本论文研究的主要内容 6

第二章 实验材料与方法 7

2.1 实验材料 7

2.1.1 主要实验试剂 7

2.1.2 主要实验仪器 7

2.2 实验方法 8

2.2.1 CRL的纯化 8

2.2.2 橄榄油乳化液的配置 8

2.2.3 酶活的测定方法 8

2.2.4 酶活计算公式 9

2.2.5 荧光光谱的测定 9

第三章 结果与讨论 10

3.1 CRL的纯化 10

3.2 酶活测定 10

3.3 荧光光谱检测 11

3.4 热力学分析 12

第四章 结论与展望 14

4.1 结论 14

4.2 展望 14

参考文献 15

致 谢 19

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