论文总字数：40038字

摘 要

戊二胺是一种重要的化学中间体，在材料、食品、医药及农业领域都具有十分重要的利用价值。L-赖氨酸脱羧酶（LDC）是生物法生产1,5-戊二胺的关键酶，研究LDC直接关系到1,5-戊二胺的工业化生产。为了开发新的L-赖氨酸脱羧酶以满足戊二胺的生产需求，本文对一种来源于Serratia proteamaculans NJ303的L-赖氨酸脱羧酶（SpLDC）进行探究。

首先对SpLDC的基因和氨基酸序列进行了分析，氨基酸序列同源性分析表明SpLDC与诱导型E. coli CadA同源性为90.21%，与E. coli LdcC同源性为69.99%，确定其为一种诱导型赖氨酸脱羧酶；对其进行同源建模以及辅酶5-磷酸吡哆醛（PLP）、底物分子L-赖氨酸（L-lys）对接研究，表明预测的其与PLP结合相关的氨基酸位点为Thr-220、Ser-221、His-245、Trp-333、Ser-364、His-366、和 Lys-367，与底物L-赖氨酸预测的作用氨基酸为Asp-330、Ser-364、Thr-304和Lys-367；将该酶编码基因SpLDC成功构建到pETDute-1质粒载体上，并导入E. coli BL21（DE3）中进行表达，并优化其表达条件，结果表明在温度为22℃，诱导剂IPTG浓度为0.05 mM，诱导时间为18 h时，表达效果最佳。

对重组酶SpLDC进行了纯化，研究了其酶学性质；结果如下：SpLDC最适催化温度和pH分别为52℃和6.5；在 pH 5.5~8.0孵育12 h酶活还能保持70%~90%，pH稳定性较好；在热稳定性研究中发现37℃~52℃孵育12 h，其酶活还能保持80%以上的催化活性，高于52℃，酶活急剧下降；在不同浓度金属离子对酶活的研究中，发现Cr³ 等离子可以促进酶活，Co² 等离子低浓度促进，高浓度抑制，Zn² 等离子对酶活都存在抑制现象；该酶的米氏常数K_m为14.45 mM，最大反应速率V_max为1525 U/mg，戊二胺抑制常数IC₅₀为61.86 mM。由此可见SpLDC具有优秀的应用前景，对于戊二胺的工业化生产具有重要意义。

关键词：L-赖氨酸脱羧酶 构建表达 结构模拟 表达优化 酶学性质

Heterologous expression and enzymatic properties of an L-lysine decarboxylase derived from Serratia proteamaculans

Abstract

Cadaverine is an important chemical intermediate and has great application value in the fields of materials, food, medicine and agriculture. L-lysine decarboxylase is a vital enzyme for the production of cadaverine by biological methods. The study of SpLDC directly relates to the industrial production of cadaverine. In order to develop new L-lysine decarboxylase to meet the production needs of cadaverine, this article explores an L-lysine decarboxylase (SpLDC) derived from Serratia proteamaculans NJ303.

First, the gene and amino acid sequence of SpLDC were analyzed. The homology analysis of amino acid sequence showed that SpLDC was 90.21% homologous to inducible E. coli CadA and 69.99% homologous to E. coli LdcC, thus confirming it to be an inducible lysine decarboxylase; In structural analysis, homology modeling using amino acid sequences and studing on the docking of pyridoxal 5-phosphate (PLP) and L-lysine (L-lys) which respectively as a coenzyme and substrate molecule. The amino acid sites binding with PLP were found to be Thr-220, Ser-221, His-245, Trp-333, Ser-364, His-366, and Lys-367. The binding sites for L-lysine are Asp-330, Ser-364, Thr-304 and Lys-367. The gene encoding the enzyme SpLDC was successfully constructed on the pETDute-1, and introduced into E. coli BL21 (DE3) for expression. The expression conditions were also optimized, and the results showed that at 22 ℃, 0.05 mM IPTG and 18 h, the expression effect is the best.

The recombinant enzyme SpLDC was purified, and its optimal catalytic temperature and pH, temperature and pH stability, the effect of metal ions on enzyme catalysis and enzyme kinetic parameters were studied. The results are as follows: The optimal catalytic temperature and pH of SpLDC are 52℃ and 6.5 respectively in the range of pH 5.5 ~ 8.0, and the enzyme activity can maintain 70% ~ 90% after 12 hours of incubation, and the pH stability is better; It was found that incubating at 37℃ ~ 52℃ for 12 h, its enzyme activity can still maintain more than 80% of its catalytic activity, and above 52℃, the enzyme activity dropped sharply; In the study of different concentrations of metal ions on enzyme activity, it was found that Cr³ can promote enzyme activity, Co² low concentration promotion, high concentration inhibition, and Zn² and other enzyme activity inhibition phenomena; The Mie constant K_m of the enzyme is 14.45 mM, the maximum reaction rate V_max is 1525 U/mg, and the IC50 of 1,5-pentanediamine inhibition constant is 61.86 mM. This shows that SpLDC has excellent application prospects and is of great significance for the industrial production of cadaverine.

Key Words: L-lysine decarboxylase; Construct expression; Structural simulation; Expression optimization; Enzymatic properties

目 录

摘要 I

ABSTRACT II

第一章 引言 1

1.1 L-赖氨酸脱羧酶简介 1

1.1.1 L-脱羧酶来源及生物学功能 1

1.1.2 L-赖氨酸脱羧酶蛋白结构研究 2

1.2 L-赖氨酸脱羧酶在生产戊二胺中的应用 3

1.2.1 1,5-戊二胺的来源 3

1.2.2 1,5-戊二胺的应用 3

1.2.3 1,5-戊二胺的生物法制备 5

1.3 本论文研究目的和意义 10

1.4 本论文研究内容 10

第二章 SpLDC的克隆表达、序列结构分析及培养条件优化 12

2.1 前言 12

2.2 材料与方法 12

2.2.1菌株与质粒 12

2.2.2培养基 13

2.2.3 SpLDC基因扩增及重组质粒构建 13

2.2.4 菌株构建正确性验证 13

2.2.5 SpLDC氨基酸序列比对及结构模拟分析 14

2.2.6 重组酶SpLDC表达条件优化 14

2.2.7 酶活检测及定义 14

2.2.8 L-赖氨酸和1,5-戊二胺检测方法 15

2.3 结果与讨论 15

2.3.1 SpLDC重组质粒的构建及结果验证 15

2.3.2 SpLDC的氨基酸序列分析及结构模拟 18

2.3.3 不同诱导温度对SpLDC表达的影响 21

2.3.4 不同诱导剂浓度对SpLDC表达的影响 21

2.3.5 不同诱导时间对 SpLDC 蛋白表达的影响 23

2.4 本章小结 23

第三章 SpLDC的酶学性质研究 25

3.1 前言 25

3.2材料与方法 25

3.2.1实验菌株 25

3.2.2 培养基 25

3.2.3 SpLDC培养表达及细胞破碎 25

3.2.4 SpLDC纯化方法 26

3.2.5 SpLDC的SDS-PAGE分析 26

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