论文总字数：21464字

摘 要

β-葡萄糖苷酶是纤维素分解酶系中的重要组成成分，在农产品加工、医药、环保方面等都得到了广泛的用途。但是β-葡萄糖苷酶的价格昂贵, 且使用寿命和贮存期较短。采用固定化技术将β-葡萄糖苷酶固定在载体上反复使用,起到工艺简化、达到降低成本的目的，还可有效提高酶的稳定性。

以SBA-15介孔分子筛为载体植被得到的固定化GLU酶活回收率可达98.1%；4℃下贮藏60天，经20次转化后，固定化酶活力还是可以保留最初的82.1%；SBA15-GLU对pNPG亲和力常数较低于游离酶；固定化酶催化pNPG水解E_a与游离酶相比也同样相应的降低；在不同温度下，SBA15-GLU的失活k_d均较游离酶低。经拟合SBA15-GLU的失活E_d相比于游离酶有显著的减小。

关键词：β-葡萄糖苷酶，固定化，SBA-15，SBA15-GLU

Immobilization of β-glucosidase on Molecular Sieve Mesoporous Silica SBA-15

Abstract

β-glucosidase, also known as gentibiose enzyme, cellobiases and amygdalin enzyme, which is an important component of cellulose-decomposing enzymes and is widely used in the processing of agricultural products, medicine, environmental protection, etc. However, the price of β-glucosidase is expensive, and the service life and storage period is short. Immobilized β-glucosidase enzyme can be used repeatedly, and the technique can simplify processes, as well as reduce the costs.

Mesoporous molecular sieve can provide a homogeneous and adjustable pore system, which is the important carrier of the immobilized enzyme.In this work, SBA-15 was used to immobilize β-glucosidase and the structure of the immobilized β-glucosidase was used to characterize the catalytic properties and stability of the grape glycosides before and after immobilization.

Determine the optimal conditions for immobilization: the yield of activity was 98.1% at the ratio of GLU/support was 29.94 U/g after incubation at room temperature for 40min. The thermal and pH stability of the immobilized GLU was comparatively higher than that in its free form. After storage at 4 ℃ for 60 days and repeatly used for 20 batches, the activity of the immobilized GLU remained 82.1% of its initial activity. The kinetic parameters of SBA15-GLU to pNPG was 1.78 mmol/L, but lower than the free enzyme (2.51 mmol/L) . The activation energy (E_a) of β- glucosidase were 11 kJ/mol and 4.76kJ/mol for free and immobilized GLU. At different temperatures, the reaction rate of deactivation (k_d) about SBA15-GLU are lower than the free enzyme.

Key words: β-glucosidase, Immobilization, SBA-15, SBA15-GLU

目 录

摘 要 I

Abstract II

第一章 文献综述 1

1.1 β-葡萄糖苷酶概述 1

1.1.1 β-葡萄糖苷酶的性质 2

1.1.2 β-葡萄糖苷酶的应用 2

1.2 酶的固定化技术 2

1.2.1 酶的固定化方法 3

1.3 分子筛SBA15材料 3

1.4 本课题的研究内容及意义 4

第二章 β-葡萄糖苷酶固定化酶的制备和表征 5

2.1 实验材料 5

2.1.1 主要实验试剂 5

2.1.2 主要实验仪器 6

2.2 试验方法 6

2.2.1 固定化酶的制备 6

2.2.2 β-葡萄糖苷酶酶活测定 7

2.2.3 蛋白浓度的测定 7

2.2.4 酶活回收率 7

2.2.5 固定化条件的研究 7

2.2.6 固定化酶的表征 8

2.3 结果与讨论 8

2.3.1 β-葡萄糖苷酶在SBA-15上的固定化条件 8

2.3.2 固定化前后载体的微观形貌分析及孔结构参数的测定 10

2.3.3 固定化前后载体的红外光谱分析 12

2.3.4 固定化前后SBA-15的X射线衍射分析 13

2.4 本章小结 14

第三章 SBA15-GLU的催化特性和稳定性分析 16

3.1 实验材料 16

3.1.1 主要实验试剂 16

3.1.2 主要实验仪器 16

3.2 实验方法 17

3.2.1 GLU酶活测定 17

3.2.2 固定化酶的制备 17

3.2.3 固定化酶的稳定性 17

3.2.4 最适作用pH 18

3.2.5 最适作用温度 18

3.2.6 GLU水解反应动力学常数的测定 18

3.2.7 GLU水解反应活化能（Ea）的测定 18

3.2.8 GLU热失活曲线与失活动力学和热力学参数的测定 18

3.3 结果与讨论 19

3.3.1 SBA15-GLU的稳定性 19

3.3.2 固定化GLU的催化特性 21

3.4 本章小结 26

第四章 结论和展望 28

4.1 结论 28

4.2 展望 29

参考文献 30

致 谢 32

第一章 文献综述

1.1 β-葡萄糖苷酶概述

β-葡萄糖苷酶（β-D-Glucosidase，EC3.2.1.21），又称为β-D-葡萄糖苷葡萄糖水解酶，它还有另外几个名字，比如龙胆二糖酶、纤维二糖酶（cellobias，CB或β-G）等，是纤维素分解酶系组成成分之一，也是很重要的一部分。它一方面能够水解最后没有还原性的β-D-葡萄糖，一方面还能生成β-D-葡萄糖以及它的配位基团。

图1-1 β-葡萄糖苷酶分子式

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