耐有机溶剂蛋白酶PT121结构稳定关键氨基酸初步分析毕业论文
2022-06-23 20:02:18
论文总字数：21673字
摘 要
由耐有机溶剂极端微生物产生的天然蛋白酶能在温和条件下展现高效的选择性和可操作性，但是由于酶作为天然生物活性大分子，在高温及有机溶剂中极易失活，因此常常需要借助一定的手段进行酶分子改造，如基于传统方法的化学修饰、固定化；基于分子生物学方法的定向进化。从某种意义上说，对于酶分子耐性机理研究将有助于提供酶分子定向进化的理论指导。
本实验室通过对Pseudomonas aeruginosa 高产菌株所产的蛋白酶进行筛选,得到蛋白酶PT121，其在高浓度亲水有机溶剂如甲醇、DMSO、DMF中能高效催化合成多种二肽，并且转化率均达到80%以上，经过克隆转换可以作为进行酶分子的耐有机溶剂分子基础研究的良好的模型。本论文从实验室筛选的耐有机溶剂蛋白酶PT121出发，通过对随机突变建立的突变库中突变体的关键突变位点进行研究，对其酶学性质进行分析，结合分子模拟等手段探讨了关键位点对酶稳定性的作用。
实验中对蛋白酶PT121进行随机突变筛选，获得11株在25%乙腈中稳定性显著变化的突变体，其中1株稳定性显示上升。通过对这11株突变体的关键突变位点进行研究及酶学性质分析，发现氨基残基改变对蛋白酶PH稳定性影响并不显著，且证实了蛋白酶温度稳定性与溶剂稳定性存在着正相关性。同时在通过对突变体的同源建模分析发现，突变位点由较小的侧链基团替换为较大的侧链时会导致蛋白酶的稳定性上升。本次所得的突变体在合成小肽研究中表明，这些突变体中大多数突变位点对小肽合成的催化效率影响较小。
关键词：耐有机溶剂蛋白酶 蛋白酶PT121 稳定性 分子动力学模拟
The preliminary analysis of the key amino acids about the structure stability of organic solvent resistant protease PT121
ABSTRACT
Natural protease produced by organic solvents resistant extremophiles exhibit high selectivity and operability under environmentally friendly conditions. However, enzyme as a natural biological active molecule, can be inactivated easily in the high temperature and in the organic solvent. For this reason, some methods should be used to remake the enzyme. For example, we can use chemical modification and immobilization that based on the conventional method, and directed evolution that based on the Molecular Biology. In a sense, the patience mechanism of enzyme molecules will provide theoretical guidance to directed evolution.
Proteases obtained by the high-yielding strains of Pseudomonas aeruginosa, which can efficient catalytic synthesis of a variety of dipeptide, and conversion rates were above 80% in the high concentration of the hydrophilic organic solvent such as methanol, DMF and DMSO. After cloning and transforming, it can be a good model of the basic research about organic solvent resistant molecules. Based on the organic solvent resistant protease PT121, random mutation was used to construct the mutated gene libraries. Then, analyzing its enzymatic properties, using molecular modeling and other means can investigate the role of key sites on the enzyme stability.
In the experiment, 11 kinds of mutants that showed significantly different stability in 25% of acetonitrile were got by random mutagenesis, followed by screening or selection, and one of which increased the stability. By researching and analyzing the key amino acids and its enzymatic properties, we found that changes in amino acid residues were not significantly relative to protease stability of PH. And it was confirmed that there is a positive correlation between the temperature stability and the solvent stability. While analyzing the mutants by homology modeling, we found that a smaller side chain groups replaced by the larger one caused which increased protease stability. What’s more, the mutants in the study of synthetic peptide showed that the most sites had little effect on the catalytic efficiency.
KEY WORDS: organic solvent resistance protease, protease PT121, stability, random mutation
目 录
摘 要 I
ABSTRACT II
第一章 文献综述 1
1.1 非水相酶概述 1
1.1.1 非水相酶促反应优势 1
1.1.2 非水相酶开发与应用 1
1.2 天然耐有机溶剂酶 2
1.2.1 来源与特性 2
1.2.2 天然耐有机溶剂蛋白酶 2
1.3 天然耐有机溶剂酶的结构机制 3
1.4 本论文研究的目的和主要内容 5
第二章 蛋白酶PT121溶剂稳定性关键氨基酸解析 6
2.1 前言 6
2.2 实验材料 7
2.2.1 菌株及质粒 7
2.2.2 实验试剂 7
2.2.3 实验仪器 8
2.2.4 培养基配方 9
2.2.5 模拟及分析软件 10
2.3 实验方法 10
2.3.1 蛋白酶PT121基因突变库的构建 10
2.3.2 突变库重组子的连接、转化、初筛及表达 11
2.3.2.1 突变库重组子的连接 11
2.3.2.1.2 双酶切反应 11
2.3.2.1.2 连接反应 11
2.3.2.2 突变库重组子的转化 12
2.3.2.2.1 制备感受态细胞 12
2.3.2.2.2转化 12
2.3.2.3 奶粉平板鉴定阳性重组子 13
2.3.2.4 突变库重组子表达酶液的快速制备 13
2.3.3 突变蛋白酶的动力学常数Km值测定 13
2.3.4 突变蛋白酶的稳定性研究 13
2.3.4.1 pH稳定性 13
2.3.4.2 温度稳定性 14
2.3.4.3 有机溶剂对突变蛋白酶稳定性的影响 14
2.3.5 典型突变体的结构分析 14
2.3.6 优势突变体在有机相中催化阿斯巴甜前体的合成 14
2.3.6.1 Z-Asp-Phe-NH2 二肽的合成条件 14
2.3.6.2 HPLC检测条件 14
2.4 结果与讨论 15
2.4.1 随机突变筛选结果 15
2.4.2 突变蛋白酶的动力学常数Km值 15
2.4.3 典型突变体的稳定性研究 16
2.4.3.1 典型突变体的pH稳定性 16
2.4.3.2 典型突变体的温度稳定性 17
2.4.3.3 有机溶剂对典型突变体蛋白酶稳定性的影响 17
2.4.4 典型突变体的结构分析 18
2.4.5 蛋白酶PT121及突变体合成阿斯巴甜前体测定结果 20
2.5 本章小结 21
第三章 结论与展望 23
3.1 结论 23
3.2 展望 23
参考文献 25
致 谢 26
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