论文总字数：24026字

摘 要

本文研究内容是关于L- 丙氨酸消旋酶的基因工程菌的建立及其发展前景。研究的基因工程菌可以通过消旋 L- 丙氨酸生产 DL- 丙氨酸，且用量少、反应时间短、可以多批次重复利用，通过本发明的基因工程菌，消旋过程的转化率达到 99.0％以上，产品纯度达到 99.5％以上，具有良好的产业化前景。

随着 L- 丙氨酸生产工艺的日臻成熟，利用 L- 丙氨酸生产高附加值衍生产品的需求与日俱增，其中DL- 丙氨酸的高效和绿色生产工艺正得到业界的重视。DL- 丙氨酸是L- 丙氨酸的结构消旋物，其化学名称是 DL-α- 氨基丙酸 ( 简称丙氨酸 )，分子式为 CH₃CH(NH₂) COOH，成品是无色至白色无臭针状结晶或结晶性粉末，有较强的甜味，易溶于水、无旋光性^[1]。 DL- 丙氨酸的用途广泛。目前主要用作食品工业中的营养强化剂和调味品，同时也是一种非常重要的医药中间体和维生素 B₆ 的生产原料，此外还可以用于新型农药的合成等技术领域。在国外，DL- 丙氨酸已经产业化，其年需求量很大，且增长迅速^[2]；当今的主要生产国为日本，主要生产企业有武藏野化学株式会社和味之素公司等。在我国，DL- 丙氨酸的生产基本采用化学合成法，例如 Strecker 法、Buchere 法、α- 卤代羧酸氨化法、相转移催化合成法以及 L- 丙氨酸化学消旋法等。但这些合成方法成本偏高，而且使用了如氰化物、甲醇、氯仿等人体危害物、副产物分离也十分困难；与此同时还暴露出合成路线长、收率低、“三废”问题严重等缺陷^[3]。

在提倡低碳经济的新政策背景下，利用生物法生产 DL- 丙氨酸的工艺在近年来得到学者们的研究。其中以L- 丙氨酸为原料、以从自然界中筛选得到的含有L- 丙氨酸消旋酶的微生物作为生物催化体，消旋 L- 丙氨酸生产 DL- 丙氨酸的工艺逐步崭露头角；该方法所需的微生物培养成本低廉、反应条件温和、生产工艺清洁，因而具有良好的应用前景。

由于基因工程技术在医学研究领域的逐渐成熟，利用分子克隆和外源表达可以更大地增加某种工业酶在宿主中的表达量，通过这种方法构建的基因工程菌株具有普通微生物难以企及的酶催化效率；利用基因工程菌株生产 DL- 丙氨酸的工艺在国内尚没有报道，本文选择一株从自然界筛选得到的大肠杆菌K-12 作为分子生物学操作的出发菌株，通过PCR 技术从该菌株的基因组上扩增得到了L- 丙氨酸消旋酶的编码基因 (alr 基因和DadX基因 )，利用大肠杆菌作为宿主，成功构建了能够高效表达 L- 丙氨酸消旋酶

的基因工程菌DM280a(Pet-28a-Alr/BL21(DE3)),DM280b(pet-28a-DadX/BL21(DE3)).通过消旋转化的DL-丙氨酸达到99.9%.

关键词：大肠杆菌 丙氨酸消旋酶 DL-丙氨酸 酶活

Preparation of DL alanine by biological method

Abstract

This paper studies a genetic engineering bacterium containing the L-alanine racemase gene and its application. The studied genetically engineered bacteria can produce DL-alanine by racemic L-alanine and have a small amount of reaction time and can be reused in multiple batches. By the genetic engineering bacteria of the present invention, the conversion of the racemic process Rate of 99.0% or more, the product purity of 99.5% or more, with good prospects for industrialization。

With the growing maturity of L-alanine production process, the use of L-alanine production of high value-added derivative products growing demand, which DL-alanine efficient and green production process is the industry's attention. DL-alanine is the structural racemate of L-alanine, its chemical name is DL-α-aminopropionic acid (abbreviated as alanine), the formula CH3CH (NH2) COOH, the product is colorless to white Stinky crystalline or crystalline powder, a strong sweet, soluble in water, no optical rotation [1]. DL-alanine is widely used. At present, it is mainly used as a nutrition enhancer and seasoning in the food industry. It is also a very important raw material for pharmaceutical intermediates and vitamin B6. It can also be used in the technical field of new pesticide synthesis. In foreign countries, DL-alanine has been industrialized, its annual demand is very large, and the rapid growth [2]; today's major producing countries for Japan, the main production enterprises are Musashino Chemical Co., Ltd. and Ajinomoto company. In our country, the production of DL-alanine is basically carried out by chemical synthesis method, such as Strecker method, Buchere method, α-halogenated carboxylic acid amination method, phase transfer catalysis synthesis method and L-alanine chemical racemate method. However, the expenditure of synthetic ways are hign, and the appliance of cyanide, methanol, chloroform and other human hazards, by-product separation is also very difficult; at the same time also exposed the synthetic route long, low yield, "three wastes" serious problems Defects [3].

In the context of the new policy of promoting low-carbon economy, the use of biological production of DL-alanine in recent years, scholars have been studied. Among them, L-alanine is

used as raw material, and the microorganism containing L-alanine racemase obtained from nature is selected as the biocatalyst, and the process of producing DL-alanine by racemic L-alanine is gradually The method requires the low cost of microbial culture, mild reaction conditions, clean production process, and thus has a good application prospects.

With the rapid development of genetic engineering technology, the use of molecular cloning and exogenous expression technology can greatly improve the expression of certain industrial enzymes in host microorganisms. The genetic engineering strains constructed by this method have the ability of ordinary microorganisms In this paper, a strain of Escherichia coli K-12, which was screened from nature, was selected as the starting strain for molecular biology operation. The strain was isolated from the strain by PCR. (Alr gene and DadX gene) were obtained from the genome of L-alanine racemase. The Escherichia coli was used as the host to construct the genetically engineered bacteria capable of efficiently expressing L-alanine racemase DM280a (Pet-28a-Alr / BL21 (DE3)), DM280b (pet-28a-DadX / BL21 (DE3)) was obtained by racemic transformation of DL-alanine to 99.9%.

Keywords: Escherichia coli alanine racemase DL-alanine enzyme activity

目 录

摘 要 I

第一章 文献综述 3

1.1 DL-丙氨酸的理化性质 3

1.2 DL-丙氨酸的应用 3

1.3 DL-丙氨酸生产方法概述 3

1.3.1化学法 3

1.3.2直接发酵法 6

1.3.3丙氨酸消旋酶法 6

1.4 DL-丙氨酸的供需现状以及发展趋势 7

1.5 产DL丙氨酸基因工程菌的研究现状 7

1.6 大肠杆菌与DL—丙氨酸合成相关的基因与酶 7

1.7 丙氨酸消旋酶酶活测定方法 8

1.8 本论文的研究目的及内容 8

第二章 实验器材和方法 10

2.1 实验材料 10

2.1.1实验仪器及试剂 10

2.1.2质粒与菌株 10

2.1.3酶与试剂 10

2.1.4培养基 10

2.2 实验方法 11

2.2.1目的基因的提取 11

2.2.2引物设计与合成 11

2.2.3丙氨酸消旋酶基因克隆 13

2.2.4表达载体的构建与转化（一步克隆法） 14

2.2.5丙氨酸消旋酶的活性测定 16

2.2.6L-丙氨酸的标准曲线测定 17

第三章 结果分析 18

3.1 丙氨酸消旋酶基因克隆 18

3.2 载体的构建与转化 18

3.3 丙氨酸消旋酶基因蛋白表达SDS-PAGE测定 19

3.4 各工程菌表达丙氨酸消旋酶活性比较 19

第四章 结论与展望 20

4.1 小结 20

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