论文总字数：27155字

摘 要

脂肪酶具有高选择性、高稳定性、反应条件温和、不需要辅酶等优良特性，能高效催化水解、酯化、转酯化、醇解、氨解等系列反应，已被广泛应用于医药、化工、食品、饲料和能源等行业。然而脂肪酶在有机溶剂、高温、极端pH等非天然环境下极易失活，这些因素极大地限制了脂肪酶在工业上的应用。枯草芽孢杆菌脂肪酶A（BSLA）是由枯草芽孢杆菌分泌产生的一种胞外水解酶，为常温酶，其最适温度仅为35 ℃。热稳定性是评价工业生物催化剂的一个重要指标，工业上提高反应温度，可以提高转化率、底物可溶性、降低微生物污染的可能性等，因此，通过分子改造提高BSLA的耐热性、使其适应工业生产过程的要求，具有重要的实际应用价值。

本实验以从RCSB数据库中获取的高分辨率BSLA晶体结构为基础，通过Pymol可视化软件与B-因子（B-factor）确定位于蛋白质表面且柔性较大区域的氨基酸位点，并基于对酶结构与功能关系的认知，分析确定与热稳定性相关的氨基酸突变位点，最终确定了以下5个高突变率残基及结果可能对BSLA热稳定性起到重要作用。再采用分子动力学模拟技术解析突变位点的引入对稳定BSLA蛋白结构的作用机制，分析BSLA与突变体BSLA^N174E的均方根偏差RMSD及二级结构(α、β)百分含量，最终确定提高其热稳定性的重要氨基酸位点是Asn174Glu。通过全质粒PCR技术构建突变株BSLA^N174E并通过实验验证突变株的热稳定性，将纯化后的BSLA和BSLA^N174E酶液分别于55℃下分别保存5、20、60 min后，突变酶BSLA^N174E相对残余活力分别是原始菌株BSLA的1.6、2.0、2.3倍。

关键词：枯草芽孢杆菌脂肪酶 B因子 热稳定性 定点突变

The site directed mutation of B- factor improves the thermal stability of BSLA

ABSTRACT

Lipase has the characteristics of high selectivity, high stability, mild reaction conditions, no coenzyme , can effectively catalyze the hydrolysis, esterification, transesterification,alcoholysis, ammonolysis reaction series, has been widely used in medicine, chemical, food, feed and energy and other industries. However, the lipase in the organic solvent, high temperature, extreme pH and other non natural environment can easily deactivation, these factors greatly limit the application of lipase in industry. Bacillus subtilis lipase A (BSLA) was a extracellular enzyme secreted by Bacillus subtilis, enzyme for normal temperature,the optimum temperature is only 35. The thermal stability is an important indicator for the evaluation of industrial catalysts, increase reaction temperature in industry, can improve the conversion rate, substrate soluble, reduce the possibility of microbial contamination, so, improving the heat resistance of BSLA by molecular transformation and adapting it to the process of industrial production, have important practical value.

This experiment is based on the high-resolution BSLA crystal structure obtained from the RCSB database, the amino acid sites on the surface of protein with high flexibility were determined by visualized software Pymol and B-factor values. Among these sites, five amino acid residues related to enzyme thermostability were finally picked out based on the cognition of the relationship between enzymes structure and function. Then molecular dynamics simulations (MDS) was performed to analyze the effects of mutations on the stability of protein structure. Asn174Glu was selected as the most important site according to the analysis of the RMSD (Root Mean Square Fluctuation) and percentage content of secondary structure (α、β) of BSLA and BSLA^N174E. Mutant strain BSLA^N174E was constructed by PCR of whole plasmid and the thermal stability of BSLA and BSLA^N174E were tested under 55℃ preservation for 5, 20 and 60 minutes. The relative residual activity of BSLA^N174E were 1.6, 2.0 and 2.3 times of BSLA respectively.

Key words:Bacillus subtilis lipase；B factor；Thermal stability；Site- directed mutagenesis

目 录

摘要 I

ABSTRACT II

第一章 文献综述 1

1.1 枯草芽孢杆菌脂肪酶A 1

1.1.1 脂肪酶概述 1

1.1.2 枯草芽孢杆菌脂肪酶A简介 1

1.2 脂肪酶的应用 3

1.3 脂肪酶热稳定性分子改造研究进展 4

1.4 分子动力学模拟 5

1.4.1 分子动力学模拟简介 5

1.4.2 分子动力学模拟技术在脂肪酶研究中的应用 5

第二章 实验材料与方法 7

2.1 实验材料 7

2.1.1 实验试剂 7

2.1.2 实验仪器 8

2.1.3 实验菌株及载体质粒 9

2.1.4 主要溶液与培养基 10

2.2 实验方法 11

2.2.1 分子模拟计算方法 11

2.2.2 重组质粒的构建 12

2.2.3 BSLA的诱导表达 14

2.2.4 BSLA的分离和纯化 14

2.2.5 相关酶学性质的测定 15

第三章 结果与讨论 17

3.1 预测BSLA突变热点区域 17

3.2 确定位于蛋白质表面且柔性较大区域的氨基酸位点 17

3.3 热稳定性相关的氨基酸位点的选取 18

3.4 模拟预测突变位点引入对BSLA蛋白稳定性的影响 19

3.5 从B. subtilis 168中获取目的基因 20

3.6 突变体的构建、表达及纯化 21

3.6.1 重组菌株的构建 21

3.6.2 全质粒PCR法定点突变 22

3.6.3 突变酶的表达及纯化 22

3.7 热稳定性测定 23

3.7.1 p-NPP标准曲线绘制 23

3.7.2 牛血清蛋白浓度标准曲线绘制 24

3.7.3 突变酶与原始酶的热稳定性分析 24

第四章 结论与展望 26

4.1 结论 26

4.2 展望 26

参考文献 27

致 谢 30

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