论文总字数：23964字

摘 要

Abstract II

第一章 绪论 1

1.1D-氨基酸简介 1

1.2D-氨基酸的发现 1

1.2.1D-氨基酸的研究进展： 2

1.2.2D-氨基酸在生物体的分布： 2

1.3D-氨基酸的生理功能 2

1.3.1D-氨基酸在调节细菌生长和代谢中的作用 2

1.3.2D-氨基酸对生物膜的形成和分散的影响 3

1.4D-氨基酸的合成和代谢 4

1.5D-氨基酸的制备方法及其进展： 5

1.5.1苯丙氨酸合成路线 5

1.5.2苯丙氨酸拆分方法 5

1.6D-氨基酰化酶的发展： 6

1.4.1D-氨基酰化酶的分类： 6

1.4.1D-氨基酰化酶的理化性质： 7

1.4.2D-氨基酰化酶的作用原理： 8

第二章 实验部分 8

2.1 实验材料 8

2.1.1菌株与质粒 8

2.1.2试剂 8

2.1.3仪器设备 9

2.2实验方法 9

2.2.1BL21/DAA工程菌的构建 9

2.2.2DAA酶的诱导表达 10

2.2.3SDS-PAGE分析 10

2.2.4凝胶的观察 10

2.3结果 11

2.3.1IPTG浓度对酶诱导表达的影响 11

2.3.2温度对酶诱导表达的影响 11

2.3.3乳糖对对酶诱导表达的影响 13

第三章 讨论 15

第四章 不足与改进 15

4.1实验中遇到的问题 15

4.1.1实验设备污染的问题 16

4.1.2制作蛋白质上样液时吹打菌体沉淀的问题 16

4.1.3制作蛋白质上样液时加热裂解的问题 16

4.1.4电泳内槽的密闭性问题 16

4.1.5外槽加入缓冲液后底部出现气泡的问题 17

4.1.6电泳缓冲液污染问题 17

4.1.7处理废液、废菌的问题 17

4.2实验的改进 17

4.2.1菌种培养阶段的改进 18

4.2.2菌种培养阶段、表达阶段时间的改进 18

4.2.3表达阶段温度的改进 18

4.2.4电泳阶段的改进 18

4.2.5凝胶的观察手段的改进 18

4.2.6量化凝胶观测结果的改进 18

参考文献 20

致 谢 22

摘 要

本文通过改变不同的实验条件，研究了D-苯丙氨酸的酶法制备中，脱乙酰基酶的表达条件优化途径。

生物酶法具有反应的高度专一性，反应条件温和，环境友好、能耗低，收率高等优点，是制备D-氨基酸的发展方向。通过对菌种给予不同的培养条件和不同的表达条件，以此反复进行对照试验，最终通过SDS-PAGE使脱乙酰基酶的表达可视化，对比蛋白条带从而确定并优化培养及表达条件。数次实验表明，在诱导表达剂为IPTG，温度恒定时，表达效率与其浓度成正比，浓度一定时，表达效率与温度成反比（30℃-37℃）；诱导表达剂为乳糖和α-乳糖时，温度恒定时，表达效率与其浓度成正比，浓度一定时，表达效率与温度成正比（30℃-37℃）；同时，在相同温度下（30℃-37℃），乳糖及α乳糖的诱导表达效率高于IPTG。

实验中，考虑到每一组实验用到的细菌可能出现偏差，且培养时间与表达时间上会存在组间差异，因此，组与组之间并不形成对照，只存在组内对照，前一组实验得出的结论进而用于设计后一组实验，从而一步一步优化出表达条件。

关键词：脱乙酰基酶，条件优化，诱导表达，SDS-PAGE

Abstract

In this paper, the optimal conditions for the expression of deacetylase in the enzymatic preparation of D-phenylalanine were studied by changing different experimental conditions.

The biological enzymatic method has the advantages of high specificity of reaction, mild reaction conditions, environmental friendliness, low energy consumption and high yield, and is a development direction for preparing D-amino acid. The control experiment was repeated by administering different culture conditions and different expression conditions to the strain, and finally the expression of the deacetylase was visualized by SDS-PAGE, and the protein bands were compared to determine and optimize the culture and expression conditions. Several experiments have shown that when the expression agent is IPTG, the expression efficiency is proportional to its concentration when the temperature is constant. When the concentration is constant, the expression efficiency is inversely proportional to the temperature (30°C-37°C); the expressionagent is lactose and α-lactose. When the temperature is constant, the expression efficiency is proportional to its concentration. When the concentration is constant, the expression efficiency is proportional to the temperature (30 ° C - 37 ° C); meanwhile, at the same temperature (30 ° C - 37 ° C), lactose and alpha lactose The induction efficiency was higher than that of IPTG.

In the experiment, it is considered that the bacteria used in each group of experiments may be biased, and there will be differences between the culture time and the expression time. Therefore, there is no control between the groups, only the intra-group control, the previous one The conclusions from the group experiments were then used to design the latter set of experiments to optimize the expression conditions step by step.

KEY WORDS: deacetylase, optimization, induced expression, SDS-PAGE

第一章 绪论

1.1D-氨基酸简介

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