论文总字数：27672字

摘 要

青霉素酰化酶在医药工业中是一种非常重要的酶，在偏碱性条件下可以制备半合成β-内酰胺抗生素所需的关键中间体，例如6-氨基青霉烷酸(6-APA)。而在酸性以及中性条件下，可用于催化合成多种新一代半合成抗生素。因其良好的催化酰化/去酰化的特性，它在工业化生产上，具有很高的应用价值。

本实验构建C端上含有6个组氨酸标签的青霉素酰化酶表达载体pET-28a/pgapx02，并将其导入E.coli BL21中诱导表达，诱导表达后酶活达到1470 U/L，比活力达到 459.38 U/L/OD₆₀₀。本实验采用Ni Sepharose Performance纯化技术来分离纯化蛋白，将过滤的酶液，先用缓冲A洗脱杂蛋白，再用缓冲B洗脱目的蛋白，纯化后的蛋白，比活力为26.56 U/mg，回收率为17.5%，纯化倍数可达83倍。利用组氨酸标签蛋白纯化的方法，能一步纯化出高纯的青霉素酰化酶，为后续进行结晶、突变等研究做了铺垫。

关键词：青霉素酰化酶 表达载体 分离纯化 组氨酸标签

Isolation and purification of key enzymes in the synthesis of antibiotics

Abstract

In the pharmaceutical industry,Penicillin acylation is a very important enzyme.Under alkaline conditions,it can be prepared semisynthetic β-lactam antibiotics to be key intermediates, such as 6-aminopenicilanic acid(6-APA).It can be used for catalyze and synthesize a variety of a new generation of semisynthetic antibiotics in acidic and neutral condition.Because of the characteristics of good catalytic acylation/deacylaction,it has the very high application value in industrial production.

The experiment is building the expression vector of penicillin acylation pET-28a/pgapx02, which contains six histidine in C-terminal, and importing into a host of E.coli BL21.Expressing induction for 24h,the enzyme activity can up to 1470 U/L,and the specific activity can up to 459.38 U/L/OD600.Using Ni Sepharose Performance technology for the separation and purification of protein.Filtered enzyme solution,with phosphate buffer A washes out impurity protein.And then,adding phosphate buffer B,the objective protein is eluted.After the purification of the protein,the specific activity is 26.56 U/mg,the recovery rate is 17.5%,and the purification fold can up to 83 times.By using the method of His-tagged protein purification,we can purify the penicillin acylase of high purity.For the subsequent research on crystallization and mutation do twisted.

Key Words:penicillin acylase; expression vector;separation and purification; His-tagged

目 录

摘要.....................................................................................................................................................Ӏ

Abstract..............................................................................................................................................ӀӀ

第一章 文献综述...........................................................................................................................1

1.1青霉素酰化酶的概述.................................................................................................................1

1.1.1青霉素酰化酶的来源..........................................................................................................1

1.1.2青霉素酰化酶的分类..........................................................................................................1

1.1.3 pH稳定性............................................................................................................................2

1.1.4热稳定性..............................................................................................................................2

1.1.5青霉素酰化酶的应用..........................................................................................................2

1.2组氨酸标签蛋白分离纯化.........................................................................................................3

1.2.1重组蛋白分离纯化的简介..................................................................................................3

1.2.2组氨酸标签蛋白分离纯化的原理......................................................................................3

1.2.3组氨酸标签蛋白分离纯化的流程......................................................................................3

1.3本课题研究意义与内容.............................................................................................................4

1.3.1研究意义..............................................................................................................................4

1.3.2研究内容..............................................................................................................................4

第二章 重组菌pET-28a/pgapx02的构建...............................................................................5

2.1前言.............................................................................................................................................5

2.2实验材料.....................................................................................................................................5

2.2.1菌株与质粒..........................................................................................................................5

2.2.2实验试剂..............................................................................................................................5

2.2.3实验仪器..............................................................................................................................6

2.2.4培养基..................................................................................................................................7

2.3实验方法.....................................................................................................................................7

2.3.1大肠杆菌E.coli BL21感受态的制备及转化.....................................................................7

2.3.2含组氨酸标签的PGA表达载体pET-28a/pgapx02的构建...............................................8

2.3.3重组菌pET-28a/pgapx02的发酵表达..............................................................................11

2.3.4 PGA酶活力的检测...........................................................................................................11

2.4结果与讨论...............................................................................................................................13

2.4.1表达载体pET-28a/pgapx02的构建..................................................................................13

2.4.2阳性克隆子的菌落PCR鉴定...........................................................................................13

2.4.3重组质粒双酶切的验证....................................................................................................14

2.5本章小结...................................................................................................................................14

第三章 PGA的分离纯化..........................................................................................................16

3.1前言...........................................................................................................................................16

3.2实验材料...................................................................................................................................16

3.2.1菌种来源............................................................................................................................16

3.2.2实验试剂............................................................................................................................16

3.2.3实验仪器............................................................................................................................16

3.3实验方法...................................................................................................................................17

3.3.1利用Ni Sepharose Performance纯化技术来纯化蛋白....................................................17

3.3.2 PGA酶活力的测定...........................................................................................................18

3.3.3 SDS-PAGE蛋白电泳........................................................................................................18

3.3.4测定蛋白含量....................................................................................................................20

3.4结果与讨论...............................................................................................................................21

3.4.1 PGApx02纯化出峰结果图...............................................................................................21

3.4.2纯化后PGApx02的SDS-PAGE分析以及结果测定.......................................................22

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