摘要精氨酸脱亚胺酶（Arginine Deiminase，简称ADI）是ADI途径精氨酸降解的第一个酶, 催化精氨酸产生瓜氨酸和氨，在工业、医学等领域具有广泛的应用前景。使用产精氨酸脱亚胺酶的基因工程菌BL21(DE3)(pET-30a-cit)，对该酶的分离纯化进行了研究。用乳糖诱导BL21(DE3)(pET-30a-cit) 精氨酸脱亚胺酶的基因表达，超声波破壁得到该酶，以包涵体和游离状态存在。包涵体经过洗涤后，以8M尿素，10mMDTT溶液为变性剂使包涵体变性溶解，采用动析方法让变性剂浓度随时间逐渐变低而变性的ADI就慢慢正确重叠。游离状态的ADI通过（NH4）2SO4盐析，DEAE-纤维素离子交换层析进行分离纯化。酶的比酶活由最初的211U/mg蛋白提高到949U/mg蛋白，纯度提高了4.5倍。活性由最初的2562U，最后收集得到1767U，活性回收率为69%。67505

毕业论文关键词：精氨酸脱亚胺酶，分离，纯化

毕业设计说明书（论文）英文摘要

Title: Purification of recombinant arginine 

deiminase in Escherichia Coli

Abstract:

ADI catalyzes the deamination of arginine to citrulline and ammonia,the first step of ADI pathway. In industry, medicine and other fields ADI has broad application prospects. In the present research the purification of ADI in E.coli BL21 which was constructed in the lab was studied. Under the induction of 0.5% lactose, ADI was expresssed in E.coli BL21(DE3),then isolated purified by breaking the cellswall. Recombinant ADI with inclusion bodis and the free state in two forms exist. After washing of inclusion bodies was dissolved in 8M urea,10mM DTT solution，then using dialysis methods refolding denatured ADI. The recombinant ADI (rADI) in the free state was purified in precipitation with （NH4）2SO4 and DEAD-cellulose ion-exchange chromatography。The ratio of enzyme activtiy of ADI was enhanced from 211U/mg to 949U/mg.The ratio of purification was 4.5 folds. The recovery of the enzyme activity was 69%.

Keyword:  Arginine deiminase  isolation  purification

目次

1  引言 1

1.1  精氨酸脱亚氨基酶（arginine deiminase,ADI)简介 1

1.1.1  精氨酸脱亚氨基酶的生物学意义 1

1.1.2  精氨酸脱亚氨基酶的理化性质 2

1.2  精氨酸脱亚氨基酶的用途 2

1.2.1  酶法转化制备L-瓜氨酸 3

1.2.2  精氨酸缺陷型癌症的治疗 3

1.2.3  其他应用 3

1.3  精氨酸脱亚胺酶的克隆表达 3

1.4  本实验室构建重组精氨酸脱亚氨基酶的酶学性质 4

1.4.1  基因工程菌BL21（DE3）（pET-30a-cit）的的介绍 4

1.4.2  BL21（DE3）（pET-30a-cit）精氨酸脱亚氨基酶的性质 5

1.5  本课题的研究内容 5

2  材料和方法 6

2.1  材料 6

2.2  精氨酸脱亚氨基酶的活性测定 8

2.3  蛋白质含量的测定 10

2.4  SDS-PAGE 电泳 11

2.5  BL21(DE3)(pET-30a-cit)的液体培养 12

2.6  精氨酸脱亚氨基酶的包涵体收集及粗酶液的提取