论文总字数：12679字

摘 要

L-丙氨酸主要用于生化研究、组织培养、医学鉴定，食品添加剂等领域。其在在医药，食品工业中有广泛的应用。工业上主要以L-天冬氨酸为原料，经脱羧、过滤、结晶干燥得到L-丙氨酸。其生产工艺的关键酶就是天冬氨酸β-脱羧酶。本研究通过基因工程的手段构建高效表达体系，提高天冬氨酸脱羧酶的活性，改善工艺路线，节约生产成本。

关键词：质粒构建 L-丙氨酸 天冬氨酸β-脱羧酶

ABSTRACT

L- alanine is mainly used in biochemical studies, tissue culture, medical identification, food additives and other fields．It is widely used in medicine and food industry.. Industrial mainly make L- aspartate as raw material, through the removal of sodium, filtration, crystallization and drying to get L- alanine. The key of the production process is the enzyme aspartate beta decarboxylase. This study building high efficient expression system，improving the activity of aspartate decarboxylase process improvement and saving production costs by means of gene engineering．

Key Words ：Plasmid construction L- alanine Aspartic acid beta decarboxylase

目录

摘要 -----------------------------------------------------------------------------------I

ABSTRACT -----------------------------------------------------I

第一章：文献综述 ----------------------------------------------------------------1

1.1L-丙氨酸的用途与生产方法 --------------------------------------------------1

1.1.1L-丙氨酸的用途 ----------------------------------------------------------------1

1.1.2L-丙氨酸的生产方法 ---------------------------------------------------------1

1.2天冬氨酸β-脱羧酶的研究进展 -----------------------------------------------1

1.3基因工程酶的表达 ---------------------------------------------------------------2

第二章：材料与方法 --------------------------------------------------------------2

2.1本实验设计路线 ----------------------------------------------------------------2

2.2. 实验 ----------------------------------------------------------------------------4

2.2.1 实验器材 ----------------------------------------------------------------------4

2.2.2 -实验试剂 ----------------------------------------------------------------------5

2.2.3质粒与菌株----------------------------------------------------------------------6

2.2.4 分子克隆操作及方法---------------------------------------------------------6

2.2.5 培养基-------------------------------------------------------------------------6

2.2.6 测序 -------------------------------------------------------------------6

2.2.7 感受态细胞的制备和转化 ---------------------------------------------------6

2.2.8质粒快检方法 -----------------------------------------------------------------7

2.2.9 测定方法-------------------------------------------------------------------------7

2.2.9.1 天冬氨酸脱酸酶酶活测定方法-----------------------------7

2.2.9.2 菌体OD的测量------------------------------------------8

第三章：结果与讨论 ---------------------------------------------------------------8

3.1基因的调取 ------------------------------------------------------------------------8

3.2 Asd基因表达载体的鉴定 -----------------------------------------------------9

3.3 Asd基因表达载体的测序鉴定 -----------------------------------------------9

3.4天冬氨酸脱羧酶酶活的初步检测 --------------------------------------------10

3.5 天冬氨酸脱羧酶菌株生产丙氨酸 -------------------------------------------11

第四章 结论与展望 -------------------------------------------------------------------12

参考文献 --------------------------------------------------------------------------------12

致谢 --------------------------------------------------------------------------------------14

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